Taci-immunoglobulin fusion proteins

ABSTRACT

Molecules that interfere with the binding of a tumor necrosis factor receptor with its ligand, such as a soluble receptor, have proven usefulness in both basic research and as therapeutics. The present invention provides improved soluble transmembrane activator and calcium modulator and cyclophilin ligand-interactor (TACI) receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/359,801, filed Jan. 26, 2009, now allowed, which is a divisional ofU.S. application Ser. No. 11/242,294, filed Oct. 3, 2005, now U.S. Pat.No. 7,501,497, which is a continuation of U.S. application Ser. No.10/152,363, now abandoned, filed May 20, 2002, which claims the benefitof U.S. Provisional Application Ser. No. 60/293,343, filed May 24, 2001,each of which are herein incorporated by reference.

TECHNICAL FIELD

The present invention relates generally to improved fusion proteinscomprising a tumor necrosis factor receptor moiety and an immunoglobulinmoiety. In particular, the present invention relates to improvedTACI-immunoglobulin fusion proteins.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 381096seqlist.txt, a creation date of Oct. 23, 2009, and a sizeof 104 Kb. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

Cytokines are soluble, small proteins that mediate a variety ofbiological effects, including the regulation of the growth anddifferentiation of many cell types (see, for example, Arai et al., Annu.Rev. Biochem. 59:783 (1990); Mosmann, Curr. Opin. Immunol. 3:311 (1991);Paul and Seder, Cell 76:241 (1994)). Proteins that constitute thecytokine group include interleukins, interferons, colony stimulatingfactors, tumor necrosis factors, and other regulatory molecules. Forexample, human interleukin-17 is a cytokine which stimulates theexpression of interleukin-6, intracellular adhesion molecule 1,interleukin-8, granulocyte macrophage colony-stimulating factor, andprostaglandin E2 expression, and plays a role in the preferentialmaturation of CD34+ hematopoietic precursors into neutrophils (Yao etal., J. Immunol. 155:5483 (1995); Fossiez et al., J. Exp. Med. 183:2593(1996)).

Receptors that bind cytokines are typically composed of one or moreintegral membrane proteins that bind the cytokine with high affinity andtransduce this binding event to the cell through the cytoplasmicportions of the certain receptor subunits. Cytokine receptors have beengrouped into several classes on the basis of similarities in theirextracellular ligand binding domains. For example, the receptor chainsresponsible for binding and/or transducing the effect of interferons aremembers of the type II cytokine receptor family, based upon acharacteristic 200 residue extracellular domain.

Cellular interactions, which occur during an immune response, areregulated by members of several families of cell surface receptors,including the tumor necrosis factor receptor (TNFR) family. The TNFRfamily consists of a number of integral membrane glycoprotein receptorsmany of which, in conjunction with their respective ligands, regulateinteractions between different hematopoietic cell lineages (see, forexample, Cosman, Stem Cells 12:440 (1994); Wajant et al., CytokineGrowth Factor Rev. 10:15 (1999); Yeh et al., Immunol. Rev. 169:283(1999); Idriss and Naismith, Microsc. Res. Tech. 50:184 (2000)).

One such receptor is TACI, transmembrane activator and CAML-interactor(von Büllow and Bram, Science 228:138 (1997); Bram and von Büllow, U.S.Pat. No. 5,969,102 (1999)). TACI is a membrane bound receptor, which hasan extracellular domain containing two cysteine-rich pseudo-repeats, atransmembrane domain and a cytoplasmic domain that interacts with CAML(calcium-modulator and cyclophilin ligand), an integral membrane proteinlocated at intracellular vesicles which is a co-inducer of NF-ATactivation when overexpressed in Jurkat cells. TACI is associated with Bcells and a subset of T cells. Nucleotide sequences that encode TACI andits corresponding amino acid sequence are provided herein as SEQ ID NOs:1 and 2, respectively

The TACI receptor binds two members of the tumor necrosis factor (TNF)ligand family. One ligand is variously designated as ZTNF4, “BAFF,”“neutrokine-α,” “BLyS,” “TALL-1,” and “THANK” (Yu et al., internationalpublication No. WO98/18921 (1998), Moore et al., Science 285:269 (1999);Mukhopadhyay et al., J. Biol. Chem. 274:15978 (1999); Schneider et al.,J. Exp. Med. 189:1747 (1999); Shu et al., J. Leukoc. Biol. 65:680(1999)). The amino acid sequence of ZTNF4 is provided as SEQ ID NO:3.The other ligand has been designated as “ZTNF2,” “APRIL” and “TNRF deathligand-1” (Hahne et al., J. Exp. Med. 188:1185 (1998); Kelly et al.,Cancer Res. 60:1021 (2000)). The amino acid sequence of ZTNF2 isprovided as SEQ ID NO:4. Both ligands are also bound by the B-cellmaturation receptor (BCMA) (Gross et al., Nature 404:995 (2000)). Thenucleotide and amino acid sequence of BCMA are provided as SEQ ID NO:26and SEQ ID NO:27, respectively.

The demonstrated in vivo activities of tumor necrosis factor receptorsillustrate the clinical potential of soluble forms of the receptor.Soluble forms of the TACI receptor have been generated as immunoglobulinfusion proteins. Initial versions resulted in low-expressing,heterogeneous protein. The heterogeneity was observed at the TACI aminoterminus, at the Fc carboxyl terminus, and in the TACI stalk region. Aneed therefore exists for pharmaceutically useful TACI receptorcompositions.

BRIEF SUMMARY OF THE INVENTION

The present invention provides improved TACI-immunoglobulin fusionproteins suitable as therapeutic compounds.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the amino acid sequence of human TACI. The locations of thecysteine-rich pseudo-repeats are indicated by shading, the transmembranedomain is boxed, and the stalk region is indicated by hash marks.

FIG. 2 is a schematic diagram of an immunoglobulin of the IgG1 subclass.C_(L): light chain constant region; C_(H1), C_(H2) C_(H3): heavy chainconstant regions; V_(L): light chain variable region; V_(H): heavy chainvariable region; CHO: carbohydrate; N: amino terminus; C: carboxylterminus.

FIGS. 3A, 3B, 3C, and 3D show a comparison of the wild-type human γ1constant region Fc amino acid sequence with variants Fc-488, Fc4, Fc5,Fc6, Fc7, and Fc8. The C_(H1) domain of the human γ1 constant region isnot part of the Fc and is therefore not shown. The location of the hingeregion, the C_(H2), and the C_(H3) domains are indicated. The Cysresidues normally involved in disulfide bonding to the light chainconstant region (LC) and heavy chain constant region (HC) are indicated.A “.” symbol indicates identity to wild-type at that position, while“***” indicates the location of the carboxyl terminus, and illustratesthe difference in the carboxyl terminus of Fc6 relative to the other Fcversions. Amino acid locations are indicated by EU index positions.

FIG. 4 shows the specific binding of ¹²⁵I-ZTNF4 with various TACI-Fcconstructs. The TACI-Fc fusion proteins had TACI moieties that lackedthe first 29 amino acid residues of the amino acid sequence of SEQ IDNO:2. One of the fusion proteins had a TACI moiety with an intact stalkregion (TACI (d1-29)-Fc5), whereas three of the TACI-Fc fusion proteinshad TACI moieties with various deletions in the stalk region (TACI(d1-29, d107-154)-Fc5; TACI (d1-29, d111-154)-Fc5; TACI (d1-29,d120-154)-Fc5). Experimental details are described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION 1. Overview

As described below, the present invention provides transmembraneactivator and calcium modulator and cyclophilin ligand-interactor(TACI)-immunoglobulin fusion proteins, and methods for usingTACI-immunoglobulin fusion proteins. For example, the present inventionprovides methods for inhibiting the proliferation of tumor cells,comprising administering to the tumor cells a composition that comprisesa TACI-immunoglobulin fusion protein. Such a composition can beadministered to cells cultured in vitro. Alternatively, the compositioncan be a pharmaceutical composition that comprises a pharmaceuticallyacceptable carrier and a TACI-immunoglobulin fusion protein, and thepharmaceutical composition can be administered to a subject, which has atumor. The subject may be a mammalian subject. Administration of thepharmaceutical composition can inhibit, for example, the proliferationof B lymphocytes in a mammalian subject.

The present invention also provides methods for inhibiting ZTNF4activity in a mammal, comprising administering to the mammal acomposition that comprises a TACI-immunoglobulin. The ZTNF4 activity canbe associated with various diseases and disorders. For example, apharmaceutical composition that comprises a TACI-immunoglobulin fusionprotein can be used to treat an autoimmune disease, such as systemiclupus erythematosus, myasthenia gravis, multiple sclerosis, insulindependent diabetes mellitus, Crohn's disease, rheumatoid arthritis,polyarticular-course juvenile rheumatoid arthritis, and psoriaticarthritis. Alternatively, a pharmaceutical composition that comprises aTACI-immunoglobulin can be used to treat a disorder such as asthma,bronchitis, emphysema, and end stage renal failure. A pharmaceuticalcomposition comprising a TACI-immunoglobulin can also be used to treatrenal disease, such as glomerulonephritis, vasculitis, nephritis,amyloidosis, and pyelonephritis, or a disorder, such as neoplasm,chronic lymphocytic leukemia, multiple myeloma, non-Hodgkin's lymphoma,post-transplantation lymphoproliferative disease, and light chaingammopathy. In certain cases, the ZTNF4 activity can be associated withT cells. A pharmaceutical composition that comprises aTACI-immunoglobulin can also be used to treat a disease or disorderassociated with immunosuppression, graft rejection, graft versus hostdisease, and inflammation. For example, a pharmaceutical compositionthat comprises a TACT-immunoglobulin can be used to decreaseinflammation, and to treat disorders such as joint pain, swelling,anemia, and septic shock.

The present invention also provides methods for reducing circulatingblood levels of ZTNF4 in a mammalian subject, comprising administeringto the mammalian subject a pharmaceutical composition that comprises apharmaceutically acceptable carrier and a TACI-immunoglobulin fusionprotein, wherein administration of the pharmaceutical compositionreduces the circulating level of ZTNF4 in the blood of the mammaliansubject. As an illustration, the administration of such a pharmaceuticalcomposition can reduce circulating blood levels of ZTNF4 by at least10%, by at least 20%, by at least 10 to 60%, by at least 20 to 50%, orby at least 30 to 40%, compared with the blood level of ZTNF4 prior tothe administration of the pharmaceutical composition. Those of skill inthe art can measure circulating levels of ZTNF4. Illustrative methodsare described in Example 4 and Example 5.

As described below, illustrative TACI-immunoglobulin fusion proteinscomprise:

-   -   (a) a TACI receptor moiety that consists of a fragment of a        polypeptide that has the amino acid sequence of amino acid        residues 30 to 154 of SEQ ID NO:2, wherein the TACI receptor        moiety comprises at least one of (i) amino acid residues 34 to        66 of SEQ ID NO:2, and (ii) amino acid residues 71 to 104 of SEQ        ID NO:2, and wherein the TACI receptor moiety binds at least one        of ZTNF2 or ZTNF4, and    -   (b) an immunoglobulin moiety comprising a constant region of an        immunoglobulin.        Suitable TACI receptor moieties include: polypeptides that        comprise amino acid residues 34 to 66 of SEQ ID NO:2, and amino        acid residues 71 to 104 of SEQ ID NO:2; polypeptides that        comprise amino acid residues 34 to 104 of SEQ ID NO:2;        polypeptides that comprise the amino acid sequence of amino acid        residues 30 to 110 of SEQ ID NO:2; and polypeptides that have an        amino acid sequence consisting of amino acid residues 30 to 110        of SEQ ID NO:2.

The immunoglobulin moiety of a TACI-immunoglobulin fusion protein cancomprise a heavy chain constant region, such as a human heavy chainconstant region. An IgG1 heavy chain constant region is one example of asuitable heavy chain constant region. An illustrative IgG1 heavy chainconstant region is an IgG1 Fc fragment that comprises C_(H2), and C_(H3)domains. The IgG1 Fc fragment can be a wild-type IgG1 Fc fragment or amutated IgG1 Fc fragment, such as the Fc fragment comprising the aminoacid sequence of SEQ ID NO:33. One exemplary TACI-immunoglobulin fusionprotein is a protein that has an amino acid sequence comprising theamino acid sequence of SEQ ID NO:54.

The TACI-immunoglobulin fusion proteins described herein can bemultimers, such as dimers.

The present invention also provides nucleic acid molecules that encode aTACI-immunoglobulin fusion protein. An illustrative nucleotide sequencethat encodes a TACI-immunoglobulin fusion protein is provided by SEQ IDNO:53.

The present invention also includes TACI soluble receptors that consistof a fragment of a polypeptide that has the amino acid sequence of aminoacid residues 30 to 154 of SEQ ID NO:2, wherein the TACI solublereceptor comprises at least one of (i) amino acid residues 34 to 66 ofSEQ ID NO:2, and (ii) amino acid residues 71 to 104 of SEQ ID NO:2, andwherein the TACI soluble receptor binds at least one of ZTNF2 or ZTNF4.Additional TACI soluble receptors are described herein as suitable TACIreceptor moieties for TACI-immunoglobulin fusion proteins. Moreover,TACI soluble receptors can be used in methods described forTACI-immunoglobulin fusion proteins.

These and other aspects of the invention will become evident uponreference to the following detailed description and drawings. Inaddition, various references are identified below and are incorporatedby reference in their entirety.

2. Definitions

In the description that follows, a number of terms are used extensively.The following definitions are provided to facilitate understanding ofthe invention.

As used herein, “nucleic acid” or “nucleic acid molecule” refers topolynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid(RNA), oligonucleotides, fragments generated by the polymerase chainreaction (PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acid molecules canbe composed of monomers that are naturally-occurring nucleotides (suchas DNA and RNA), or analogs of naturally-occurring nucleotides (e.g.,α-enantiomeric forms of naturally-occurring nucleotides), or acombination of both. Modified nucleotides can have alterations in sugarmoieties and/or in pyrimidine or purine base moieties. Sugarmodifications include, for example, replacement of one or more hydroxylgroups with halogens, alkyl groups, amines, and azido groups, or sugarscan be functionalized as ethers or esters. Moreover, the entire sugarmoiety can be replaced with sterically and electronically similarstructures, such as aza-sugars and carbocyclic sugar analogs. Examplesof modifications in a base moiety include alkylated purines andpyrimidines, acylated purines or pyrimidines, or other well-knownheterocyclic substitutes. Nucleic acid monomers can be linked byphosphodiester bonds or analogs of such linkages. Analogs ofphosphodiester linkages include phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like. The term “nucleic acidmolecule” also includes so-called “peptide nucleic acids,” whichcomprise naturally-occurring or modified nucleic acid bases attached toa polyamide backbone. Nucleic acids can be either single stranded ordouble stranded.

The term “complement of a nucleic acid molecule” refers to a nucleicacid molecule having a complementary nucleotide sequence and reverseorientation as compared to a reference nucleotide sequence. For example,the sequence 5′ ATGCACGGG 3′ (SEQ ID NO:57) is complementary to 5′CCCGTGCAT 3′ (SEQ ID NO:58).

The term “contig” denotes a nucleic acid molecule that has a contiguousstretch of identical or complementary sequence to another nucleic acidmolecule. Contiguous sequences are said to “overlap” a given stretch ofa nucleic acid molecule either in their entirety or along a partialstretch of the nucleic acid molecule.

The term “degenerate nucleotide sequence” denotes a sequence ofnucleotides that includes one or more degenerate codons as compared to areference nucleic acid molecule that encodes a polypeptide. Degeneratecodons contain different triplets of nucleotides, but encode the sameamino acid residue (i.e., GAU and GAC triplets each encode Asp).

The term “structural gene” refers to a nucleic acid molecule that istranscribed into messenger RNA (mRNA), which is then translated into asequence of amino acids characteristic of a specific polypeptide.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a growth factor that has been separated from thegenomic DNA of a cell is an isolated DNA molecule. Another example of anisolated nucleic acid molecule is a chemically-synthesized nucleic acidmolecule that is not integrated in the genome of an organism. A nucleicacid molecule that has been isolated from a particular species issmaller than the complete DNA molecule of a chromosome from thatspecies.

A “nucleic acid molecule construct” is a nucleic acid molecule, eithersingle- or double-stranded, that has been modified through humanintervention to contain segments of nucleic acid combined and juxtaposedin an arrangement not existing in nature.

“Linear DNA” denotes non-circular DNA molecules having free 5′ and 3′ends. Linear DNA can be prepared from closed circular DNA molecules,such as plasmids, by enzymatic digestion or physical disruption.

“Complementary DNA (cDNA)” is a single-stranded DNA molecule that isformed from an mRNA template by the enzyme reverse transcriptase.Typically, a primer complementary to portions of mRNA is employed forthe initiation of reverse transcription. Those skilled in the art alsouse the term “cDNA” to refer to a double-stranded DNA moleculeconsisting of such a single-stranded DNA molecule and its complementaryDNA strand. The term “cDNA” also refers to a clone of a cDNA moleculesynthesized from an RNA template.

A “promoter” is a nucleotide sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ non-codingregion of a gene, proximal to the transcriptional start site of astructural gene. Sequence elements within promoters that function in theinitiation of transcription are often characterized by consensusnucleotide sequences. These promoter elements include RNA polymerasebinding sites, TATA sequences, CAAT sequences, differentiation-specificelements (DSEs; McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclicAMP response elements (CREs), serum response elements (SREs; Treisman,Seminars in Cancer Biol. 1:47 (1990)), glucocorticoid response elements(GREs), and binding sites for other transcription factors, such asCRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye etal., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response elementbinding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamerfactors (see, in general, Watson et al., eds., Molecular Biology of theGene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), andLemaigre and Rousseau, Biochem. J. 303:1 (1994)). If a promoter is aninducible promoter, then the rate of transcription increases in responseto an inducing agent. In contrast, the rate of transcription is notregulated by an inducing agent if the promoter is a constitutivepromoter. Repressible promoters are also known.

A “core promoter” contains essential nucleotide sequences for promoterfunction, including the TATA box and start of transcription. By thisdefinition, a core promoter may or may not have detectable activity inthe absence of specific sequences that may enhance the activity orconfer tissue specific activity.

A “regulatory element” is a nucleotide sequence that modulates theactivity of a core promoter. For example, a regulatory element maycontain a nucleotide sequence that binds with cellular factors enablingtranscription exclusively or preferentially in particular cells,tissues, or organelles. These types of regulatory elements are normallyassociated with genes that are expressed in a “cell-specific,”“tissue-specific,” or “organelle-specific” manner.

An “enhancer” is a type of regulatory element that can increase theefficiency of transcription, regardless of the distance or orientationof the enhancer relative to the start site of transcription.

“Heterologous DNA” refers to a DNA molecule, or a population of DNAmolecules, that does not exist naturally within a given host cell. DNAmolecules heterologous to a particular host cell may contain DNA derivedfrom the host cell species (i.e., endogenous DNA) so long as that hostDNA is combined with non-host DNA (i.e., exogenous DNA). For example, aDNA molecule containing a non-host DNA segment encoding a polypeptideoperably linked to a host DNA segment comprising a transcriptionpromoter is considered to be a heterologous DNA molecule. Conversely, aheterologous DNA molecule can comprise an endogenous gene operablylinked with an exogenous promoter. As another illustration, a DNAmolecule comprising a gene derived from a wild-type cell is consideredto be heterologous DNA if that DNA molecule is introduced into a mutantcell that lacks the wild-type gene.

A “polypeptide” is a polymer of amino acid residues joined by peptidebonds, whether produced naturally or synthetically. Polypeptides of lessthan about 10 amino acid residues are commonly referred to as“peptides.”

A “protein” is a macromolecule comprising one or more polypeptidechains. A protein may also comprise non-peptidic components, such ascarbohydrate groups. Carbohydrates and other non-peptidic substituentsmay be added to a protein by the cell in which the protein is produced,and will vary with the type of cell. Proteins are defined herein interms of their amino acid backbone structures; substituents such ascarbohydrate groups are generally not specified, but may be presentnonetheless.

A peptide or polypeptide encoded by a non-host DNA molecule is a“heterologous” peptide or polypeptide.

An “integrated genetic element” is a segment of DNA that has beenincorporated into a chromosome of a host cell after that element isintroduced into the cell through human manipulation. Within the presentinvention, integrated genetic elements are most commonly derived fromlinearized plasmids that are introduced into the cells byelectroporation or other techniques. Integrated genetic elements arepassed from the original host cell to its progeny.

A “cloning vector” is a nucleic acid molecule, such as a plasmid,cosmid, or bacteriophage, which has the capability of replicatingautonomously in a host cell. Cloning vectors typically contain one or asmall number of restriction endonuclease recognition sites that allowinsertion of a nucleic acid molecule in a determinable fashion withoutloss of an essential biological function of the vector, as well asnucleotide sequences encoding a marker gene that is suitable for use inthe identification and selection of cells transformed with the cloningvector. Marker genes typically include genes that provide tetracyclineresistance or ampicillin resistance.

An “expression vector” is a nucleic acid molecule encoding a gene thatis expressed in a host cell. Typically, an expression vector comprises atranscription promoter, a gene, and a transcription terminator. Geneexpression is usually placed under the control of a promoter, and such agene is said to be “operably linked to” the promoter. Similarly, aregulatory element and a core promoter are operably linked if theregulatory element modulates the activity of the core promoter.

A “recombinant host” is a cell that contains a heterologous nucleic acidmolecule, such as a cloning vector or expression vector. In the presentcontext, an example of a recombinant host is a cell that produces aTACI-Fc fusion protein from an expression vector.

“Integrative transformants” are recombinant host cells, in whichheterologous DNA has become integrated into the genomic DNA of thecells.

A “fusion protein” is a hybrid protein expressed by a nucleic acidmolecule comprising nucleotide sequences of at least two genes. Forexample, a TACT-immunoglobulin fusion protein comprises a TACI receptormoiety and an immunoglobulin moiety. As used herein, a “TACI receptormoiety” is a portion of the extracellular domain of the TACI receptorthat binds at least one of ZTNF2 or ZTNF4. The phrase an “immunoglobulinmoiety” refers to a polypeptide that comprises a constant region of animmunoglobulin. For example, the immunoglobulin moiety can comprise aheavy chain constant region. The term “TACI-Fc” fusion protein refers toa TACI-immunoglobulin fusion protein in which the immunoglobulin moietycomprises immunoglobulin heavy chain constant regions, C_(H2) andC_(H3).

The term “receptor” denotes a cell-associated protein that binds to abioactive molecule termed a “ligand.” This interaction mediates theeffect of the ligand on the cell. In the context of TACI receptorbinding, the phrase “specifically binds” or “specific binding” refers tothe ability of the ligand to competitively bind with the receptor. Forexample, ZTNF4 specifically binds with the TACI receptor, and this canbe shown by observing competition for the TACI receptor betweendetectably labeled ZTNF4 and unlabeled ZTNF4.

Receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g.,thyroid stimulating hormone receptor, beta-adrenergic receptor) ormultimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor,GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6receptor). Membrane-bound receptors are characterized by a multi-domainstructure comprising an extracellular ligand-binding domain and anintracellular effector domain that is typically involved in signaltransduction. In certain membrane-bound receptors, the extracellularligand-binding domain and the intracellular effector domain are locatedin separate polypeptides that comprise the complete functional receptor.

In general, the binding of ligand to receptor results in aconformational change in the receptor that causes an interaction betweenthe effector domain and other molecule(s) in the cell, which in turnleads to an alteration in the metabolism of the cell. Metabolic eventsthat are often linked to receptor-ligand interactions include genetranscription, phosphorylation, dephosphorylation, increases in cyclicAMP production, mobilization of cellular calcium, mobilization ofmembrane lipids, cell adhesion, hydrolysis of inositol lipids andhydrolysis of phospholipids.

The term “secretory signal sequence” denotes a DNA sequence that encodesa peptide (a “secretory peptide”) that, as a component of a largerpolypeptide, directs the larger polypeptide through a secretory pathwayof a cell in which it is synthesized. The larger polypeptide is commonlycleaved to remove the secretory peptide during transit through thesecretory pathway.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature.Typically, a preparation of isolated polypeptide contains thepolypeptide in a highly purified form, i.e., at least about 80% pure, atleast about 90% pure, at least about 95% pure, greater than 95% pure, orgreater than 99% pure. One way to show that a particular proteinpreparation contains an isolated polypeptide is by the appearance of asingle band following sodium dodecyl sulfate (SDS)-polyacrylamide gelelectrophoresis of the protein preparation and Coomassie Brilliant Bluestaining of the gel. However, the term “isolated” does not exclude thepresence of the same polypeptide in alternative physical forms, such asdimers or alternatively glycosylated or derivatized forms.

The terms “amino-terminal” and “carboxyl-terminal” are used herein todenote positions within polypeptides. Where the context allows, theseterms are used with reference to a particular sequence or portion of apolypeptide to denote proximity or relative position. For example, acertain sequence positioned carboxyl-terminal to a reference sequencewithin a polypeptide is located proximal to the carboxyl terminus of thereference sequence, but is not necessarily at the carboxyl terminus ofthe complete polypeptide.

The term “expression” refers to the biosynthesis of a gene product. Forexample, in the case of a structural gene, expression involvestranscription of the structural gene into mRNA and the translation ofmRNA into one or more polypeptides.

The term “splice variant” is used herein to denote alternative forms ofRNA transcribed from a gene. Splice variation arises naturally throughuse of alternative splicing sites within a transcribed RNA molecule, orless commonly between separately transcribed RNA molecules, and mayresult in several mRNAs transcribed from the same gene. Splice variantsmay encode polypeptides having altered amino acid sequence. The termsplice variant is also used herein to denote a polypeptide encoded by asplice variant of an mRNA transcribed from a gene.

As used herein, the term “immunomodulator” includes cytokines, stem cellgrowth factors, lymphotoxins, co-stimulatory molecules, hematopoieticfactors, and synthetic analogs of these molecules.

The term “complement/anti-complement pair” denotes non-identicalmoieties that form a non-covalently associated, stable pair underappropriate conditions. For instance, biotin and avidin (orstreptavidin) are prototypical members of a complement/anti-complementpair. Other exemplary complement/anti-complement pairs includereceptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs,sense/antisense polynucleotide pairs, and the like. Where subsequentdissociation of the complement/anti-complement pair is desirable, thecomplement/anti-complement pair preferably has a binding affinity lessthan 10⁹ M⁻¹.

An “antibody fragment” is a portion of an antibody such as F(ab′)₂,F(ab)₂, Fab′, Fab, and the like. Regardless of structure, an antibodyfragment binds with the same antigen that is recognized by the intactantibody.

The term “antibody fragment” also includes a synthetic or a geneticallyengineered polypeptide that binds to a specific antigen, such aspolypeptides consisting of the light chain variable region, “Fv”fragments consisting of the variable regions of the heavy and lightchains, recombinant single chain polypeptide molecules in which lightand heavy variable regions are connected by a peptide linker (“scFvproteins”), and minimal recognition units consisting of the amino acidresidues that mimic the hypervariable region.

A “chimeric antibody” is a recombinant protein that contains thevariable domains and complementary determining regions derived from arodent antibody, while the remainder of the antibody molecule is derivedfrom a human antibody.

“Humanized antibodies” are recombinant proteins in which murinecomplementarity determining regions of a monoclonal antibody have beentransferred from heavy and light variable chains of the murineimmunoglobulin into a human variable domain.

As used herein, a “therapeutic agent” is a molecule or atom, which isconjugated to an antibody moiety to produce a conjugate, which is usefulfor therapy. Examples of therapeutic agents include drugs, toxins,immunomodulators, chelators, boron compounds, photoactive agents ordyes, and radioisotopes.

A “detectable label” is a molecule or atom, which can be conjugated toan antibody moiety to produce a molecule useful for diagnosis. Examplesof detectable labels include chelators, photoactive agents,radioisotopes, fluorescent agents, paramagnetic ions, or other markermoieties.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2:95 (1991). DNA molecules encoding affinity tags areavailable from commercial suppliers (e.g., Pharmacia Biotech,Piscataway, N.J.).

A “naked antibody” is an entire antibody, as opposed to an antibodyfragment, which is not conjugated with a therapeutic agent. Nakedantibodies include both polyclonal and monoclonal antibodies, as well ascertain recombinant antibodies, such as chimeric and humanizedantibodies.

As used herein, the term “antibody component” includes both an entireantibody and an antibody fragment.

An “immunoconjugate” is a conjugate of an antibody component with atherapeutic agent or a detectable label.

A “target polypeptide” or a “target peptide” is an amino acid sequencethat comprises at least one epitope, and that is expressed on a targetcell, such as a tumor cell, or a cell that carries an infectious agentantigen. T cells recognize peptide epitopes presented by a majorhistocompatibility complex molecule to a target polypeptide or targetpeptide and typically lyse the target cell or recruit other immune cellsto the site of the target cell, thereby killing the target cell.

An “antigenic peptide” is a peptide, which will bind a majorhistocompatibility complex molecule to form an MHC-peptide complex,which is recognized by a T cell, thereby inducing a cytotoxic lymphocyteresponse upon presentation to the T cell. Thus, antigenic peptides arecapable of binding to an appropriate major histocompatibility complexmolecule and inducing a cytotoxic T cells response, such as cell lysisor specific cytokine release against the target cell, which binds orexpresses the antigen. The antigenic peptide can be bound in the contextof a class I or class II major histocompatibility complex molecule, onan antigen presenting cell or on a target cell.

In eukaryotes, RNA polymerase II catalyzes the transcription of astructural gene to produce mRNA. A nucleic acid molecule can be designedto contain an RNA polymerase II template in which the RNA transcript hasa sequence that is complementary to that of a specific mRNA. The RNAtranscript is termed an “anti-sense RNA” and a nucleic acid moleculethat encodes the anti-sense RNA is termed an “anti-sense gene.”Anti-sense RNA molecules are capable of binding to mRNA molecules,resulting in an inhibition of mRNA translation.

Due to the imprecision of standard analytical methods, molecular weightsand lengths of polymers are understood to be approximate values. Whensuch a value is expressed as “about” X or “approximately” X, the statedvalue of X will be understood to be accurate to ±10%.

3. Production of Nucleic Acid Molecules Encoding TACI-ImmunoglobulinProteins

FIG. 1 provides the predicted amino acid sequence of human TACI (vonBillow and Bram, Science 278:138 (1997)). The TACI polypeptide containsthe following predicted elements: (a) two cysteine-rich pseudo-repeatstructures characteristic of tumor necrosis factor ligand bindingdomains, (b) a 62 amino acid “stalk region,” which resides between theligand binding domains and the transmembrane domain, (c) a 20 amino acidtransmembrane domain, and (d) a 127 amino acid intracellular domain. Theamino acid sequence does not contain a predicted hydrophobic aminoterminal signal sequence.

In order to create a soluble form of human TACI for use as an inhibitorof the native ligand:native receptor interaction, a TACI extracellulardomain—human immunoglobulin Fc fusion protein was generated. Theavailable human TACI sequence was used as the starting point fordesigning the fusion protein molecule (von Billow and Bram, Science278:138 (1997)). This initial construct, designated as “TACI-Fc4,”included amino acid residues 1 through 154 of the TACI polypeptide, anda modified human Fc region, described below. The fusion point of residue154 was chosen in order to include as much of the stalk region of TACIas possible while not including any potential portion of the predictedtransmembrane domain.

Since native TACI polypeptide does not contain an amino terminal signalsequence, an amino terminal signal sequence was added to TACI in orderto generate a secreted form of the TACI-Fc fusion protein. The signalsequence was a modified pre-pro sequence from human tissue plasminogenactivator. The modifications were included to enhance signal peptidasecleavage and furin protease-specific processing and for that reason thissequence has been referred to as the “optimized tPA (otPA) leader.” TheotPA sequence (SEQ ID NO:25) is illustrated below; modified amino acidresidues are shaded. The recombinant TACI-Fc fusion protein codingsequence was inserted into an expression vector, which was transfectedinto Chinese hamster ovary cells.

Transfected Chinese hamster ovary cells produced the TACI-Fc4 protein ata low level of about 0.3 pg/cell/day. Western blot analysis of TACI-Fcprotein with goat anti-human IgG Fc antisera revealed two bands, oneband was smaller than the expected size of approximately 48 kDa. Aminoacid sequence analysis of purified proteins revealed that the smallerband reflected cleavage of TACI fusion proteins at various sites withinthe TACI stalk region. With reference to SEQ ID NO:2, the major terminiwere found at amino acid residues 118 and 123, although, proteins werealso cleaved at amino acid positions 110, 139, and 141.

In addition to heterogeneity caused by cleavage in the stalk region,heterogeneity was also observed at the amino and carboxyl termini. Withreference to SEQ ID NO:2, the major amino termini were found at aminoacid residues 1, 10, and 13. Differences in the carboxyl terminusreflect the natural heterogeneity of recombinant immunoglobulins andimmunoglobulin fusion proteins, which includes the incomplete removal ofthe carboxyl-terminally-encoded lysine residue. Another source ofheterogeneity was found in the variable nature of the carbohydratestructure attached to the Fc encoded immunoglobulin C_(H2) domain.

New versions of TACI-Fc were generated to address the observedheterogeneity. Constructs were designed that included at least one ofthe following variations in the TACI moiety: (1) portions of the TACIstalk region were deleted, (2) a portion of the TACI stalk region wasreplaced with a portion of the BCMA stalk region, (3) the arginineresidue at position 119 was mutated to eliminate a potential furincleavage site, (4) the glutamine residue at position 121 was mutated toeliminate a potential furin cleavage site, (5) the arginine residue atposition 122 was mutated to eliminate a potential furin cleavage site,(6) amino acid residue at positions 123 and 142 were mutated to aminoacid residues found in corresponding positions of murine TACI, (7) thehuman otPA signal sequence was replaced with a human heavy chainvariable region signal sequence, (8) the valine residue at position 29was mutated to methionine, and the otPA signal sequence was joined in anamino terminal position to this residue, and (9) the otPA signalsequence was joined in an amino terminal location to the alanine residueat position 30.

Modifications were also introduced in the immunoglobulin moiety. Fiveclasses of immunoglobulin, IgG, IgA, IgM, IgD, and IgE, have beenidentified in higher vertebrates. IgG, IgD, and IgE proteins arecharacteristically disulfide linked heterotetramers consisting of twoidentical heavy chains and two identical light chains. Typically, IgM isfound as a pentamer of a tetramer, whereas IgA occurs as a dimer of atetramer.

IgG comprises the major class as it normally exists as the second mostabundant protein found in plasma. In humans, IgG consists of foursubclasses, designated IgG1, IgG2, IgG3, and IgG4. As shown in FIG. 2,each immunoglobulin heavy chain possesses a constant region thatconsists of constant region protein domains (C_(H1), hinge, C_(H2), andC_(H3)) that are invariant for a given subclass. The heavy chainconstant regions of the IgG class are identified with the Greek symbolγ. For example, immunoglobulins of the IgG1 subclass contain a yl heavychain constant region.

The Fc fragment, or Fc domain, consists of the disulfide linked heavychain hinge regions, C_(H2), and C_(H3) domains. In immunoglobulinfusion proteins, Fc domains of the IgG1 subclass are often used as theimmunoglobulin moiety, because IgG1 has the longest serum half-life ofany of the serum proteins. Lengthy serum half-life can be a desirableprotein characteristic for animal studies and potential humantherapeutic use. In addition, the IgG1 subclass possesses the strongestability to carry out antibody mediated effector functions. The primaryeffector function that may be most useful in an immunoglobulin fusionprotein is the ability for an IgG1 antibody to mediate antibodydependent cellular cytotoxicity. On the other hand, this could be anundesirable function for a fusion protein that functions primarily as anantagonist. Several of the specific amino acid residues that areimportant for antibody constant region-mediated activity in the IgG1subclass have been identified. Inclusion or exclusion of these specificamino acids therefore allows for inclusion or exclusion of specificimmunoglobulin constant region-mediated activity.

Six versions of a modified human IgG1 Fc were generated for creating Fcfusion proteins. Fc-488 was designed for convenient cloning of a fusionprotein containing the human γ1 Fc region, and it was constructed usingthe wild-type human immunoglobulin γ1 constant region as a template.Concern about potential deleterious effects due to an unpaired cysteineresidue led to the decision to replace the cysteine (amino acid residue24 of SEQ ID NO:6) that normally disulfide bonds with the immunoglobulinlight chain constant region with a serine residue. An additional changewas introduced at the codon encoding EU index position 218 (amino acidresidue 22 of SEQ ID NO:6) to introduce a BglII restriction enzymerecognition site for ease of future DNA manipulations. These changeswere introduced into the PCR product encoded on the PCR primers. Due tothe location of the BglII site and in order to complete the Fc hingeregion, codons for EU index positions 216 and 217 (amino acid residues20 and 21 of SEQ ID NO:6) were incorporated in the fusion proteinpartner sequences.

Fc4, Fc5, and Fc6 contain mutations to reduce effector functionsmediated by the Fc by reducing FcγRI binding and complement C1q binding.Fc4 contains the same amino acid substitutions that were introduced intoFc-488. Additional amino acid substitutions were introduced to reducepotential Fc mediated effector functions. Specifically, three amino acidsubstitutions were introduced to reduce FcγRI binding. These are thesubstitutions at EU index positions 234, 235, and 237 (amino acidresidues 38, 39, and 41 of SEQ ID NO:6). Substitutions at thesepositions have been shown to reduce binding to FcγRI (Duncan et al.,Nature 332:563 (1988)). These amino acid substitutions may also reduceFcγRIIa binding, as well as FcγRIII binding (Sondermann et al., Nature406:267 (2000); Wines et al., J. Immunol. 164:5313 (2000)).

Several groups have described the relevance of EU index positions 330and 331 (amino acid residues 134 and 135 of SEQ ID NO:6) in complementC1q binding and subsequent complement fixation (Canfield and Morrison,J. Exp. Med. 173:1483 (1991); Tao et al., J. Exp. Med. 178:661 (1993)).Amino acid substitutions at these positions were introduced in Fc4 toreduce complement fixation. The C_(H3) domain of Fc4 is identical tothat found in the corresponding wild-type polypeptide, except for thestop codon, which was changed from TGA to TAA to eliminate a potentialdam methylation site when the cloned DNA is grown in dam plus strains ofE. coli.

In Fc5, the arginine residue at EU index position 218 was mutated backto a lysine, because the BglII cloning scheme was not used in fusionproteins containing this particular Fc. The remainder of the Fc5sequence matches the above description for Fc4.

Fc6 is identical to Fc5 except that the carboxyl terminal lysine codonhas been eliminated. The C-terminal lysine of mature immunoglobulins isoften removed from mature immunoglobulins post-translationally prior tosecretion from B-cells, or removed during serum circulation.Consequently, the C-terminal lysine residue is typically not found oncirculating antibodies. As in Fc4 and Fc5 above, the stop codon in theFc6 sequence was changed to TAA.

Fc7 is identical to the wild-type yl Fc except for an amino acidsubstitution at EU index position 297 located in the C_(H2) domain. EUindex position Asn-297 (amino acid residue 101 of SEQ ID NO:6) is a siteof N-linked carbohydrate attachment. N-linked carbohydrate introduces apotential source of variability in a recombinantly expressed protein dueto potential batch-to-batch variations in the carbohydrate structure. Inan attempt to eliminate this potential variability, Asn-297 was mutatedto a glutamine residue to prevent the attachment of N-linkedcarbohydrate at that residue position. The carbohydrate at residue 297is also involved in Fc binding to the FcγRIII (Sondermann et al., Nature406:267 (2000)). Therefore, removal of the carbohydrate should decreasebinding of recombinant Fc7 containing fusion proteins to the FcγRs ingeneral. As above, the stop codon in the Fc7 sequence was mutated toTAA.

Fc8 is identical to the wild-type immunoglobulin γ1 region shown in SEQID NO:6, except that the cysteine residue at EU index position 220(amino acid residue 24 of SEQ ID NO:6) was replaced with a serineresidue. This mutation eliminated the cysteine residue that normallydisulfide bonds with the immunoglobulin light chain constant region.

Illustrative TACI-Fc constructs are described in Table 1.

TABLE 1 Illustrative TACI-Fc Fusion Protein Constructs TACI Sequence^(a)Fc Version TACI^(b) Fc4 TACI^(b) Fc5 TACI^(b) Fcγ1 TACI (d107-154) Fc5TACI (R119Q) Fc4 TACI (1-104)-BCMA (42-54)^(c) Fc5 TACI (d143-150) Fc5TACI (R142G, d143-150) Fc5 TACI (R119G, Q121P, R122Q, S123A) Fc5TACI(R119G, R122Q) Fc5 TACI (d1-28, V29M) Fc6 TACI (d1-29) Fc6 TACI(d1-29) Fc5 TACI (d1-29, d107-154) Fc5 TACI (d1-29, d111-154) Fc5 TACI(d1-29, d120-154) Fc5 ^(a)Information about locations, mutations, anddeletions of amino acid sequences is provided within parentheses inreference to the amino acid sequence of SEQ ID NO: 2. ^(b)Includes aminoacid residues 1 to 154 of SEQ ID NO: 2. ^(c)This construct includesamino acid residues 1 to 104 of SEQ ID NO: 2 (TACI) and amino acids 42to 54 of SEQ ID NO: 27 (BCMA).

The TACI-Fc proteins were produced by recombinant Chinese hamster ovarycells, isolated, and analyzed using Western blot analysis and amino acidsequence analysis. Surprisingly, deletion of the first 29 amino acidsfrom the N-terminus of the TACI polypeptide resulted in a ten-foldincrease in the production of TACI-Fc fusion proteins by Chinese hamsterovary cells. This deletion also reduced the cleavage of the full-lengthstalk region. In addition, cleavage within the TACI stalk region wassuppressed either by truncating the TACI stalk region, or by replacingthe TACI stalk region within another amino acid sequence (e.g., theamino acid sequence of the BCMA stalk region).

As described in Example 4, functional analyses of TACI-Fc constructsindicate that fusion proteins TACI (d1-29)-Fc5, TACI (d1-29,d107-154)-Fc5, TACI (d1-29, d111-154)-Fc5, and TACI (d1-29,d120-154)-Fc5 have similar binding affinities for ZTNF4. However,constructs, TACI (d1-29)-Fc5, TACI (d1-29, d111-154)-Fc5, and TACI(d1-29, d120-154)-Fc5 appear to bind more ZTNF4 per mole of TACI-Fc thanconstruct, TACI (d1-29, d107-154)-Fc5. Depending upon the intended use(i.e., therapeutic, diagnostic, or research), either high capacity orlow capacity TACI-Fc fusion proteins can be employed. In addition, acombination of high capacity and low capacity TACI-Fc fusion proteinsenables the titration of ZTNF2 or ZTNF4.

The present invention contemplates TACI-immunoglobulin fusion proteinsthat comprise a TACI receptor moiety consisting of amino acid residues30 to 106 of SEQ ID NO:2, 30 to 110 of SEQ ID NO:2, 30 to 119 of SEQ IDNO:2, or 30 to 154 of SEQ ID NO:2. The present invention also includesTACI-immunoglobulin fusion proteins that comprise a TACI receptor moietyconsisting of amino acid residues 31 to 106 of SEQ ID NO:2, 31 to 110 ofSEQ ID NO:2, 31 to 119 of SEQ ID NO:2, or 31 to 154 of SEQ ID NO:2.

More generally, the present invention includes TACI-immunoglobulinfusion proteins, wherein the TACI receptor moiety consists of a fragmentof amino acid residues 30 to 154 of SEQ ID NO:2, and wherein the TACIreceptor moiety binds at least one of ZTNF2 or ZTNF4. Such fragmentscomprise a cysteine-rich pseudo-repeat region, and optionally, caninclude at least one of an N-terminal segment, which resides in anamino-terminal position to the cysteine-rich pseudo-repeat region, and astalk segment, which resides in a carboxyl-terminal position to thecysteine-rich pseudo-repeat region. Suitable cysteine-rich pseudo-repeatregions include polypeptides that: (a) comprise at least one of aminoacid residues 34 to 66 of SEQ ID NO:2, and amino acid residues 71 to 104of SEQ ID NO:2, (b) comprise both amino acid residues 34 to 66 of SEQ IDNO:2, and amino acid residues 71 to 104 of SEQ ID NO:2, or (c) compriseamino acid residues 34 to 104 of SEQ ID NO:2.

Suitable N-terminal segments include the following with reference to SEQID NO:2: amino acid residue 33, amino acid residues 32 to 33, amino acidresidues 31 to 33, and amino acid residues 30 to 33. Suitable stalksegments include one or more amino acids of amino acid residues 105 to154 of SEQ ID NO:2. For example, the stalk segment can consist of thefollowing with reference to SEQ ID NO:2: amino acid residue 105, aminoacid residues 105 to 106, amino acid residues 105 to 107, amino acidresidues 105 to 108, amino acid residues 105 to 109, amino acid residues105 to 110, amino acid residues 105 to 111, amino acid residues 105 to112, amino acid residues 105 to 113, amino acid residues 105 to 114,amino acid residues 105 to 115, amino acid residues 105 to 116, aminoacid residues 105 to 117, amino acid residues 105 to 118, amino acidresidues 105 to 119, amino acid residues 105 to 120, amino acid residues105 to 121, amino acid residues 105 to 122, amino acid residues 105 to123, amino acid residues 105 to 124, amino acid residues 105 to 125,amino acid residues 105 to 126, amino acid residues 105 to 127, aminoacid residues 105 to 128, amino acid residues 105 to 129, amino acidresidues 105 to 130, amino acid residues 105 to 131, amino acid residues105 to 132, amino acid residues 105 to 133, amino acid residues 105 to134, amino acid residues 105 to 135, amino acid residues 105 to 136,amino acid residues 105 to 137, amino acid residues 105 to 138, aminoacid residues 105 to 139, amino acid residues 105 to 140, amino acidresidues 105 to 141, amino acid residues 105 to 142, amino acid residues105 to 143, amino acid residues 105 to 144, amino acid residues 105 to145, amino acid residues 105 to 146, amino acid residues 105 to 147,amino acid residues 105 to 148, amino acid residues 105 to 149, aminoacid residues 105 to 150, amino acid residues 105 to 151, amino acidresidues 105 to 152, amino acid residues 105 to 153, and amino acidresidues 105 to 154.

Additional suitable stalk segments include one or more amino acids ofthe BCMA stalk region (i.e., amino acid residues 42 to 54 of SEQ IDNO:27. For example, a stalk segment can consist of the following withreference to SEQ ID NO:27: amino acid residue 42, amino acid residues 42to 43, amino acid residues 42 to 44, amino acid residues 42 to 45, aminoacid residues 42 to 46, amino acid residues 42 to 47, amino acidresidues 42 to 48, amino acid residues 42 to 49, amino acid residues 42to 50, amino acid residues 42 to 51, amino acid residues 42 to 52, aminoacid residues 42 to 53, and amino acid residues 42 to 54. Moregenerally, a stalk segment can consist of two to 50 amino acid residues.

The immunoglobulin moiety of a fusion protein described herein comprisesat least one constant region of an immunoglobulin. Preferably, theimmunoglobulin moiety represents a segment of a human immunoglobulin.The human immunoglobulin sequence can be a wild-type amino acidsequence, or a modified wild-type amino acid sequence, which has atleast one of the amino acid mutations discussed above.

The human immunoglobulin amino acid sequence can also vary fromwild-type by having one or more mutations characteristic of a knownallotypic determinant. Table 2 shows the allotypic determinants of thehuman IgGγ1 constant region (Putman, The Plasma Proteins, Vol. V, pages49 to 140 (Academic Press, Inc. 1987)). EU index positions 214, 356,358, and 431 define the known IgGγ1 allotypes. Position 214 is in theC_(H1) domain of the IgGγ1 constant region, and, therefore, does notreside within the Fc sequence. The wild-type Fc sequence of SEQ ID NO:6includes the G1m(1) and G1m(2-) allotypes. However, the Fc moiety of aTACI-Fc protein can be modified to reflect any combination of theseallotypes.

TABLE 2 Allotypic Determinants of the Human Immunoglobulin γ1 ConstantRegion Amino Acid Amino Acid Position Allotype Residue EU Index SEQ IDNO: 6 G1m(1) Asp, Leu 356, 358 160, 162 G1m(1-) Glu, Met 356, 358 160,162 G1m(2) Gly 431 235 G1m(2-) Ala 431 235 G1m(3) Arg 214 — G1m(3-) Lys214 —

The examples of TACI-Fc proteins disclosed herein comprise human IgG1constant regions. However, suitable immunoglobulin moieties also includepolypeptides comprising at least one constant region, such as a heavychain constant region from any of the following immunoglobulins: IgG2,IgG3, IgG4, IgA1, IgA2, IgD, IgE, and IgM. Advantageously,immunoglobulin moieties derived from wild-type IgG2 or wild-type IgG4offer reduced effector function, compared with wild-type IgG1 orwild-type IgG3. The present invention also contemplates fusion proteinsthat comprise a TACI receptor moiety, as described above, and eitheralbumin or β2-macroglobulin.

Another type of receptor fusion protein that binds ZTNF2 or ZTNF4 is aBCMA-immunoglobulin fusion protein. Studies have been performed with aBCMA-Fc4 fusion protein in which the BCMA moiety consists of amino acidresidues 1 to 48 of SEQ ID NO:27. Surprisingly, pharmacokinetic studiesin mice revealed that BCMA-Fc4 fusion protein had a half-life of about101 hours, whereas a TACI-Fc protein had a half-life of 25 hours. Thus,administration of a BCMA-immunoglobulin fusion protein may be preferredin certain clinical settings. Moreover, a combination ofTACI-immunoglobulin and BCMA-immunoglobulin fusion proteins may beadvantageous to treat certain conditions. This combination therapy canbe achieved by administering TACI-immunoglobulin and BCMA-immunoglobulinfusion proteins, or by administering heterodimers of TACI-immunoglobulinand BCMA-immunoglobulin fusion proteins.

Another type of receptor fusion protein that binds ZTNF4 is animmunoglobulin fusion protein comprising an extracellular domain of areceptor designated as “Ztnfr12.” Ztnfr12 amino acid and nucleotidesequences are provided as SEQ ID NO:59 and SEQ ID NO:60, respectively.Suitable Ztnfr12 receptor moieties include polypeptides comprising aminoacid residues 1 to 69 of SEQ ID NO:60, or amino acid residues 19 to 35of SEQ ID NO:60.

The fusion proteins of the present invention can have the form of singlechain polypeptides, dimers, trimers, or multiples of dimers or trimers.Dimers can be homodimers or heterodimers, and trimers can be homotrimersor heterotrimers. Examples of heterodimers include a TACI-immunoglobulinpolypeptide with a BCMA-immunoglobulin polypeptide, aTACI-immunoglobulin polypeptide with a Ztnfr12-immunoglobulinpolypeptide, and a BCMA-immunoglobulin polypeptide with aZtnfr12-immunoglobulin polypeptide. Examples of heterotrimers include aTACI-immunoglobulin polypeptide with two BCMA-immunoglobulinpolypeptides, a TACI-immunoglobulin polypeptide with twoZtnfr12-immunoglobulin polypeptides, a BCMA-immunoglobulin polypeptidewith two Ztnfr12-immunoglobulin polypeptides, two TACI-immunoglobulinpolypeptides with a BCMA-immunoglobulin polypeptide, twoTACI-immunoglobulin polypeptides with a Ztnfr12-immunoglobulinpolypeptide, two BCMA-immunoglobulin polypeptides with aZtnfr12-immunoglobulin polypeptide, and a trimer of aTACI-immunoglobulin polypeptide, a BCMA-immunoglobulin polypeptide, anda Ztnfr12-immunoglobulin polypeptide.

In such fusion proteins, the TACI receptor moiety can comprise at leastone of the following amino acid sequences of SEQ ID NO:2: amino acidresidues 30 to 154, amino acid residues 34 to 66, amino acid residues 71to 104, amino acid residues 47 to 62, and amino acid residues 86 to 100.The BCMA receptor moiety can comprise at least one of the followingamino acid sequences of SEQ ID NO:27: amino acid residues 1 to 48, aminoacid residues 8 to 41, and amino acid residues 21 to 37. The Ztnfr12receptor moiety can comprise at least one of the following amino acidsequences of SEQ ID NO:60: amino acid residues 1 to 69, and amino acidresidues 19 to 35.

Fusion proteins can be produced using the PCR methods used to constructthe illustrative TACI-Fc molecules, which are described in the Examples.However, those of skill in the art can use other standard approaches.For example, nucleic acid molecules encoding TACI, BCMA, Ztnfr12, orimmunoglobulin polypeptides can be obtained by screening human cDNA orgenomic libraries using polynucleotide probes based upon sequencesdisclosed herein. These techniques are standard and well-established(see, for example, Ausubel et al. (eds.), Short Protocols in MolecularBiology, 3^(rd) Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995)(“Ausubel (1995)”); Wu et al., Methods in Gene Biotechnology, pages33-41 (CRC Press, Inc. 1997) (“Wu (1997)”); Ausubel (1995) at pages 5-1to 5-6; Wu (1997) at pages 307-327)).

Alternatively, molecules for constructing immunoglobulin fusion proteinscan be obtained by synthesizing nucleic acid molecules using mutuallypriming long oligonucleotides and the nucleotide sequences describedherein (see, for example, Ausubel (1995) at pages 8-8 to 8-9).Established techniques using the polymerase chain reaction provide theability to synthesize DNA molecules at least two kilobases in length(Adang et al., Plant Molec. Biol. 21:1131 (1993), Bambot et al., PCRMethods and Applications 2:266 (1993), Dillon et al., “Use of thePolymerase Chain Reaction for the Rapid Construction of SyntheticGenes,” in Methods in Molecular Biology, Vol. 15: PCR Protocols: CurrentMethods and Applications, White (ed.), pages 263-268, (Humana Press,Inc. 1993), and Holowachuk et al., PCR Methods Appl. 4:299 (1995)).

The nucleic acid molecules of the present invention can also besynthesized with “gene machines” using protocols such as thephosphoramidite method. If chemically-synthesized double stranded DNA isrequired for an application such as the synthesis of a gene or a genefragment, then each complementary strand is made separately. Theproduction of short genes (60 to 80 base pairs) is technicallystraightforward and can be accomplished by synthesizing thecomplementary strands and then annealing them. For the production oflonger genes (>300 base pairs), however, special strategies may berequired, because the coupling efficiency of each cycle during chemicalDNA synthesis is seldom 100%. To overcome this problem, synthetic genes(double-stranded) are assembled in modular form from single-strandedfragments that are from 20 to 100 nucleotides in length. For reviews onpolynucleotide synthesis, see, for example, Glick and Pasternak,Molecular Biotechnology, Principles and Applications of Recombinant DNA(ASM Press 1994), Itakura et al., Annu. Rev. Biochem. 53:323 (1984), andClimie et al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).

4. Production of TACI-Immunoglobulin Polypeptides

The polypeptides of the present invention can be produced in recombinanthost cells following conventional techniques. To express aTACI-immunoglobulin-encoding sequence, a nucleic acid molecule encodingthe polypeptide must be operably linked to regulatory sequences thatcontrol transcriptional expression in an expression vector and then,introduced into a host cell. In addition to transcriptional regulatorysequences, such as promoters and enhancers, expression vectors caninclude translational regulatory sequences and a marker gene, which issuitable for selection of cells that carry the expression vector.

Expression vectors that are suitable for production of a foreign proteinin eukaryotic cells typically contain (1) prokaryotic DNA elementscoding for a bacterial replication origin and an antibiotic resistancemarker to provide for the growth and selection of the expression vectorin a bacterial host; (2) eukaryotic DNA elements that control initiationof transcription, such as a promoter; and (3) DNA elements that controlthe processing of transcripts, such as a transcriptiontermination/polyadenylation sequence.

Expression vectors can also include nucleotide sequences encoding asecretory sequence that directs the heterologous polypeptide into thesecretory pathway of a host cell. For example, an expression vector maycomprise a nucleotide sequence that encodes TACI-immunoglobulin and asecretory sequence derived from any secreted gene. As discussed above,one suitable signal sequence is a tPA signal sequence. An exemplary tPAsignal sequence is provided by SEQ ID NO:25. Another suitable signalsequence is a murine 26-10 V_(H) signal sequence. The murine 26-10antibody is described, for example, by Near et al., Mol. Immunol. 27:901(1990). Illustrative amino acid and nucleotide sequences of a murine26-10 V_(H) signal sequence are provided by SEQ ID NO:61 and SEQ IDNO:65, respectively. SEQ ID NO:62 discloses the amino acid sequence of aTACI-Fc5 fusion protein that comprises a murine 26-10 V_(H) signalsequence.

TACI-immunoglobulin proteins of the present invention may be expressedin mammalian cells. Examples of suitable mammalian host cells includeAfrican green monkey kidney cells (Vero; ATCC CRL 1587), human embryonickidney cells (293-HEK; ATCC CRL 1573), baby hamster kidney cells(BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells(MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1; ATCC CCL61;CHO DG44 (Chasin et al., Som. Cell. Molec. Genet. 12:555, 1986)), ratpituitary cells (GH1; ATCC CCL82), HeLa S3 cells (ATCC CCL2.2), rathepatoma cells (H-4-II-E; ATCC CRL 1548) SV40-transformed monkey kidneycells (COS-1; ATCC CRL 1650) and murine embryonic cells (NIH-3T3; ATCCCRL 1658).

For a mammalian host, the transcriptional and translational regulatorysignals may be derived from viral sources, such as adenovirus, bovinepapilloma virus, simian virus, or the like, in which the regulatorysignals are associated with a particular gene which has a high level ofexpression. Suitable transcriptional and translational regulatorysequences also can be obtained from mammalian genes, such as actin,collagen, myosin, and metallothionein genes.

Transcriptional regulatory sequences include a promoter regionsufficient to direct the initiation of RNA synthesis. Suitableeukaryotic promoters include the promoter of the mouse metallothionein Igene (Hamer et al., J. Molec. Appl. Genet. 1:273 (1982)), the TKpromoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 earlypromoter (Benoist et al., Nature 290:304 (1981)), the Rous sarcoma viruspromoter (Gorman et al., Proc. Nat'l Acad. Sci. USA 79:6777 (1982)), thecytomegalovirus promoter (Foecking et al., Gene 45:101 (1980)), and themouse mammary tumor virus promoter (see, generally, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163-181 (John Wiley & Sons, Inc. 1996)). One useful combination ofa promoter and enhancer is provided by a myeloproliferative sarcomavirus promoter and a human cytomegalovirus enhancer.

Alternatively, a prokaryotic promoter, such as the bacteriophage T3 RNApolymerase promoter, can be used to control production ofTACI-immunoglobulin proteins in mammalian cells if the prokaryoticpromoter is regulated by a eukaryotic promoter (Zhou et al., Mol. Cell.Biol. 10:4529 (1990), and Kaufman et al., Nucl. Acids Res. 19:4485(1991)).

An expression vector can be introduced into host cells using a varietyof standard techniques including calcium phosphate transfection,liposome-mediated transfection, microprojectile-mediated delivery,electroporation, and the like. The transfected cells can be selected andpropagated to provide recombinant host cells that comprise theexpression vector stably integrated in the host cell genome. Techniquesfor introducing vectors into eukaryotic cells and techniques forselecting such stable transformants using a dominant selectable markerare described, for example, by Ausubel (1995) and by Murray (ed.), GeneTransfer and Expression Protocols (Humana Press 1991).

For example, one suitable selectable marker is a gene that providesresistance to the antibiotic neomycin. In this case, selection iscarried out in the presence of a neomycin-type drug, such as G-418 orthe like. Selection systems can also be used to increase the expressionlevel of the gene of interest, a process referred to as “amplification.”Amplification is carried out by culturing transfectants in the presenceof a low level of the selective agent and then increasing the amount ofselective agent to select for cells that produce high levels of theproducts of the introduced genes. A suitable amplifiable selectablemarker is dihydrofolate reductase, which confers resistance tomethotrexate. Other drug resistance genes (e.g., hygromycin resistance,multi-drug resistance, puromycin acetyltransferase) can also be used.Alternatively, markers that introduce an altered phenotype, such asgreen fluorescent protein, or cell surface proteins such as CD4, CD8,Class I MHC, placental alkaline phosphatase may be used to sorttransfected cells from untransfected cells by such means as FACS sortingor magnetic bead separation technology.

TACI-immunoglobulin polypeptides can also be produced by culturedmammalian cells using a viral delivery system. Exemplary viruses forthis purpose include adenovirus, herpesvirus, vaccinia virus andadeno-associated virus (AAV). Adenovirus, a double-stranded DNA virus,is currently the best studied gene transfer vector for delivery ofheterologous nucleic acid (for a review, see Becker et al., Meth. CellBiol. 43:161 (1994), and Douglas and Curiel, Science & Medicine 4:44(1997)). Advantages of the adenovirus system include the accommodationof relatively large DNA inserts, the ability to grow to high-titer, theability to infect a broad range of mammalian cell types, and flexibilitythat allows use with a large number of available vectors containingdifferent promoters.

By deleting portions of the adenovirus genome, larger inserts (up to 7kb) of heterologous DNA can be accommodated. These inserts can beincorporated into the viral DNA by direct ligation or by homologousrecombination with a co-transfected plasmid. An option is to delete theessential E1 gene from the viral vector, which results in the inabilityto replicate unless the E1 gene is provided by the host cell. Adenovirusvector-infected human 293 cells (ATCC Nos. CRL-1573, 45504, 45505), forexample, can be grown as adherent cells or in suspension culture atrelatively high cell density to produce significant amounts of protein(see Gamier et al., Cytotechnol. 15:145 (1994)).

Those of skill in the art can devise suitable expression vectors forproducing the fusion proteins described herein with mammalian cells.Example 4 describes features of one expression vector. As anotherexample, an expression vector can comprise a bicistronic expressioncassette that includes a portion of the human cytomegalovirus enhancer,the myeloproliferative sarcoma virus promoter, a nucleotide sequenceencoding a fusion protein, the poliovirus internal ribosomal entrysites, a nucleotide sequence encoding murine dihydrofolate reductase,followed by the SV40 poly A addition sequence. The nucleotide sequenceof SEQ ID NO:69 shows a cytomegalovirus enhancer/myeloproliferativesarcoma virus LTR promoter construct, in which the cytomegalovirusenhancer extends from nucleotide 1 to 407. The myeloproliferativesarcoma virus LTR promoter, absent the negative control region extendsfrom nucleotide 408 to nucleotide 884 of SEQ ID NO:69. A nucleotidesequence for the myeloproliferative sarcoma virus LTR promoter withoutthe negative control region is provided in SEQ ID NO:70.

Example 1 describes an expression vector that comprises acytomegalovirus promoter to direct the expression of the recombinantprotein transgene, an immunoglobulin intron, and a tissue plasminogenactivator signal sequence. One suitable immunoglobulin intron is amurine 26-10 V_(H) intron. SEQ ID NO:66 provides an illustrativenucleotide sequence of a murine 26-10 V_(H) intron. An expression vectormay also include a 5′ untranslated region (UTR) located upstream of thenucleotide sequence that encodes a TACI-immunoglobulin protein. Asuitable 5′-UTR can be derived from the murine 26-10 V_(H) gene. SEQ IDNO:63 discloses the nucleotide sequence of a useful native murine 26-10V_(H) 5′-UTR, while SEQ ID NO:64 shows the nucleotide sequence of amurine 26-10 V_(H) 5′-UTR, which has been optimized at the 3′ end.

As an illustration, SEQ ID NO:67 provides a nucleotide sequence thatincludes the following elements: a native murine 26-10 V_(H) 5′-UTR(nucleotides 1 to 51), a murine 26-10 V_(H) signal sequence (nucleotides52 to 97, and 182 to 192), a murine 26-10 V_(H) intron (nucleotides 98to 181), a nucleotide sequence that encodes a TACI moiety (nucleotides193 to 435), and a nucleotide sequence that encodes an Fc5 moiety(nucleotides 436 to 1131). The nucleotide sequence of SEQ ID NO:68differs from SEQ ID NO:67 due to the replacement of an optimized murine26-10 V_(H) 5′-UTR (nucleotides 1 to 51) for the native sequence.

TACI-immunoglobulin proteins can also be expressed in other highereukaryotic cells, such as avian, fungal, insect, yeast, or plant cells.The baculovirus system provides an efficient means to introduce clonedgenes into insect cells. Suitable expression vectors are based upon theAutographa californica multiple nuclear polyhedrosis virus (AcMNPV), andcontain well-known promoters such as Drosophila heat shock protein (hsp)70 promoter, Autographa californica nuclear polyhedrosis virusimmediate-early gene promoter (ie-1) and the delayed early 39K promoter,baculovirus p10 promoter, and the Drosophila metallothionein promoter. Asecond method of making recombinant baculovirus utilizes atransposon-based system described by Luckow (Luckow, et al., J. Virol.67:4566 (1993)). This system, which utilizes transfer vectors, is soldin the BAC-to-BAC kit (Life Technologies, Rockville, Md.). This systemutilizes a transfer vector, PFASTBAC (Life Technologies) containing aTn7 transposon to move the DNA encoding the TACI-immunoglobulinpolypeptide into a baculovirus genome maintained in E. coli as a largeplasmid called a “bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk, and Rapoport, J. Biol. Chem. 270:1543 (1995). In addition,transfer vectors can include an in-frame fusion with DNA encoding anepitope tag at the C- or N-terminus of the expressed TACI-immunoglobulinpolypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al.,Proc. Nat'l Acad. Sci. 82:7952 (1985)). Using a technique known in theart, a transfer vector containing a nucleotide sequence that encodes aTACI-immunoglobulin protein is transformed into E. coli, and screenedfor bacmids, which contain an interrupted lacZ gene indicative ofrecombinant baculovirus. The bacmid DNA containing the recombinantbaculovirus genome is then isolated using common techniques.

The illustrative PFASTBAC vector can be modified to a considerabledegree. For example, the polyhedrin promoter can be removed andsubstituted with the baculovirus basic protein promoter (also known asPcor, p6.9 or MP promoter) which is expressed earlier in the baculovirusinfection, and has been shown to be advantageous for expressing secretedproteins (see, for example, Hill-Perkins and Possee, J. Gen. Virol.71:971 (1990), Bonning, et al., J. Gen. Virol. 75:1551 (1994), andChazenbalk and Rapoport, J. Biol. Chem. 270:1543 (1995). In suchtransfer vector constructs, a short or long version of the basic proteinpromoter can be used. Moreover, transfer vectors can be constructed,with secretory signal sequences derived from insect proteins. Forexample, a secretory signal sequence from EcdysteroidGlucosyltransferase (EGT), honey bee Melittin (Invitrogen Corporation;Carlsbad, Calif.), or baculovirus gp67 (PharMingen: San Diego, Calif.)can be used in such constructs.

The recombinant virus or bacmid is used to transfect host cells.Suitable insect host cells include cell lines derived from IPLB-Sf-21, aSpodoptera frugiperda pupal ovarian cell line, such as Sf9 (ATCC CRL1711), Sf21AE, and Sf21 (Invitrogen Corporation; San Diego, Calif.), aswell as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line(Invitrogen) derived from Trichoplusia ni (U.S. Pat. No. 5,300,435).Commercially available serum-free media can be used to grow and tomaintain the cells. Suitable media are Sf900 II™ (Life Technologies) orESF 921™ (Expression Systems) for the Sf9 cells; and Ex-cellO405™ (JRHBiosciences, Lenexa, Kans.) or Express FiveO™ (Life Technologies) forthe T. ni cells. When recombinant virus is used, the cells are typicallygrown up from an inoculation density of approximately 2-5×10⁵ cells to adensity of 1-2×10⁶ cells at which time a recombinant viral stock isadded at a multiplicity of infection (MOI) of 0.1 to 10, more typicallynear 3.

Established techniques for producing recombinant proteins in baculovirussystems are provided by Bailey et al., “Manipulation of BaculovirusVectors,” in Methods in Molecular Biology, Volume 7: Gene Transfer andExpression Protocols, Murray (ed.), pages 147-168 (The Humana Press,Inc. 1991), by Patel et al., “The baculovirus expression system,” in DNACloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages205-244 (Oxford University Press 1995), by Ausubel (1995) at pages 16-37to 16-57, by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995), and by Lucknow, “Insect Cell ExpressionTechnology,” in Protein Engineering: Principles and Practice, Cleland etal. (eds.), pages 183-218 (John Wiley & Sons, Inc. 1996).

Fungal cells, including yeast cells, can also be used to express thegenes described herein. Yeast species of particular interest in thisregard include Saccharomyces cerevisiae, Pichia pastoris, and Pichiamethanolica. Suitable promoters for expression in yeast includepromoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH(alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinoldehydrogenase), and the like. Many yeast cloning vectors have beendesigned and are readily available. A vector can be designed to generateconstructs utilizing the necessary elements to carry out homologousrecombination in yeast (see, for example, Raymond et al., BioTechniques26:134 (1999)). For example, such an expression vector can include URA3and CEN-ARS (autonomously replicating sequence) sequences required forselection and replication in S. cerevisiae. Other suitable vectorsinclude YIp-based vectors, such as YIp5, YRp vectors, such as YRp17, YEpvectors such as YEp13 and YCp vectors, such as YCp19. Methods fortransforming S. cerevisiae cells with exogenous DNA and producingrecombinant polypeptides from these cells are disclosed by, for example,Kawasaki, U.S. Pat. No. 4,599,311, Kawasaki et al., U.S. Pat. No.4,931,373, Brake, U.S. Pat. No. 4,870,008, Welch et al., U.S. Pat. No.5,037,743, and Murray et al., U.S. Pat. No. 4,845,075. Transformed cellsare selected by phenotype determined by the selectable marker, commonlydrug resistance or the ability to grow in the absence of a particularnutrient (e.g., leucine). A suitable vector system for use inSaccharomyces cerevisiae is the POT1 vector system disclosed by Kawasakiet al. (U.S. Pat. No. 4,931,373), which allows transformed cells to beselected by growth in glucose-containing media. Additional suitablepromoters and terminators for use in yeast include those from glycolyticenzyme genes (see, e.g., Kawasaki, U.S. Pat. No. 4,599,311, Kingsman etal., U.S. Pat. No. 4,615,974, and Bitter, U.S. Pat. No. 4,977,092) andalcohol dehydrogenase genes. See also U.S. Pat. Nos. 4,990,446,5,063,154, 5,139,936, and 4,661,454.

Transformation systems for other yeasts, including Hansenula polymorpha,Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces fragilis,Ustilago maydis, Pichia pastoris, Pichia methanolica, Pichiaguillermondii and Candida maltosa are known in the art. See, forexample, Gleeson et al., J. Gen. Microbiol. 132:3459 (1986), and Cregg,U.S. Pat. No. 4,882,279. Aspergillus cells may be utilized according tothe methods of McKnight et al., U.S. Pat. No. 4,935,349. Methods fortransforming Acremonium chrysogenum are disclosed by Sumino et al., U.S.Pat. No. 5,162,228. Methods for transforming Neurospora are disclosed byLambowitz, U.S. Pat. No. 4,486,533.

For example, the use of Pichia methanolica as host for the production ofrecombinant proteins is disclosed by Raymond, U.S. Pat. No. 5,716,808,Raymond, U.S. Pat. No. 5,736,383, Raymond et al., Yeast 14:11-23 (1998),and in international publication Nos. WO 97/17450, WO 97/17451, WO98/02536, and WO 98/02565. DNA molecules for use in transforming P.methanolica will commonly be prepared as double-stranded, circularplasmids, which are preferably linearized prior to transformation. Forpolypeptide production in P. methanolica, the promoter and terminator inthe plasmid can be that of a P. methanolica gene, such as a P.methanolica alcohol utilization gene (AUG1 or AUG2). Other usefulpromoters include those of the dihydroxyacetone synthase (DHAS), formatedehydrogenase (FMD), and catalase (CAT) genes. To facilitate integrationof the DNA into the host chromosome, it is preferred to have the entireexpression segment of the plasmid flanked at both ends by host DNAsequences. A suitable selectable marker for use in Pichia methanolica isa P. methanolica ADE2 gene, which encodesphosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), andwhich allows ade2 host cells to grow in the absence of adenine. Forlarge-scale, industrial processes where it is desirable to minimize theuse of methanol, host cells can be used in which both methanolutilization genes (AUG1 and AUG2) are deleted. For production ofsecreted proteins, host cells can be deficient in vacuolar proteasegenes (PEP4 and PRB1). Electroporation is used to facilitate theintroduction of a plasmid containing DNA encoding a polypeptide ofinterest into P. methanolica cells. P. methanolica cells can betransformed by electroporation using an exponentially decaying, pulsedelectric field having a field strength of from 2.5 to 4.5 kV/cm,preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40milliseconds, most preferably about 20 milliseconds.

Expression vectors can also be introduced into plant protoplasts, intactplant tissues, or isolated plant cells. Methods for introducingexpression vectors into plant tissue include the direct infection orco-cultivation of plant tissue with Agrobacterium tumefaciens,microprojectile-mediated delivery, DNA injection, electroporation, andthe like. See, for example, Horsch et al., Science 227:1229 (1985),Klein et al., Biotechnology 10:268 (1992), and Miki et al., “Proceduresfor Introducing Foreign DNA into Plants,” in Methods in Plant MolecularBiology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,1993).

Alternatively, TACI-immunoglobulin proteins can be produced inprokaryotic host cells. Suitable promoters that can be used to produceTACI-immunoglobulin polypeptides in a prokaryotic host are well-known tothose of skill in the art and include promoters capable of recognizingthe T4, T3, Sp6 and T7 polymerases, the P_(R) and P_(L) promoters ofbacteriophage lambda, the trp, recA, heat shock, lacUV5, tac,lpp-lacSpr,phoA, and lacZ promoters of E. coli, promoters of B.subtilis, the promoters of the bacteriophages of Bacillus, Streptomycespromoters, the int promoter of bacteriophage lambda, the bla promoter ofpBR322, and the CAT promoter of the chloramphenicol acetyl transferasegene. Prokaryotic promoters have been reviewed by Glick, J. Ind.Microbiol. 1:277 (1987), Watson et al., Molecular Biology of the Gene,4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (1995).

Suitable prokaryotic hosts include E. coli and Bacillus subtilus.Suitable strains of E. coli include BL21(DE3), BL21(DE3)pLysS,BL21(DE3)pLysE, DH1, DH41, DH5, DH5I, DH5IF′, DH5IMCR, DH10B, DH10B/p3,DH11S, C600, HB101, JM101, JM105, JM109, JM110, K38, RR1, Y1088, Y1089,CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), MolecularBiology Labfax (Academic Press 1991)). Suitable strains of Bacillussubtilus include BR151, YB886, MI119, MI120, and B170 (see, for example,Hardy, “Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach,Glover (ed.) (IRL Press 1985)).

When expressing a TACI-immunoglobulin protein in bacteria such as E.coli, the polypeptide may be retained in the cytoplasm, typically asinsoluble granules, or may be directed to the periplasmic space by abacterial secretion sequence. In the former case, the cells are lysed,and the granules are recovered and denatured using, for example,guanidine isothiocyanate or urea. The denatured polypeptide can then berefolded and dimerized by diluting the denaturant, such as by dialysisagainst a solution of urea and a combination of reduced and oxidizedglutathione, followed by dialysis against a buffered saline solution. Inthe latter case, the polypeptide can be recovered from the periplasmicspace in a soluble and functional form by disrupting the cells (by, forexample, sonication or osmotic shock) to release the contents of theperiplasmic space and recovering the protein, thereby obviating the needfor denaturation and refolding.

Methods for expressing proteins in prokaryotic hosts are well-known tothose of skill in the art (see, for example, Williams et al.,“Expression of foreign proteins in E. coli using plasmid vectors andpurification of specific polyclonal antibodies,” in DNA Cloning 2:Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (OxfordUniversity Press 1995), Ward et al., “Genetic Manipulation andExpression of Antibodies,” in Monoclonal Antibodies: Principles andApplications, page 137 (Wiley-Liss, Inc. 1995), and Georgiou,“Expression of Proteins in Bacteria,” in Protein Engineering: Principlesand Practice, Cleland et al. (eds.), page 101 (John Wiley & Sons, Inc.1996)).

Standard methods for introducing expression vectors into bacterial,yeast, insect, and plant cells are provided, for example, by Ausubel(1995).

General methods for expressing and recovering foreign protein producedby a mammalian cell system are provided by, for example, Etcheverry,“Expression of Engineered Proteins in Mammalian Cell Culture,” inProtein Engineering: Principles and Practice, Cleland et al. (eds.),pages 163 (Wiley-Liss, Inc. 1996). Standard techniques for recoveringprotein produced by a bacterial system is provided by, for example,Grisshammer et al., “Purification of over-produced proteins from E. colicells,” in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al.(eds.), pages 59-92 (Oxford University Press 1995). Established methodsfor isolating recombinant proteins from a baculovirus system aredescribed by Richardson (ed.), Baculovirus Expression Protocols (TheHumana Press, Inc. 1995).

As an alternative, polypeptides of the present invention can besynthesized by exclusive solid phase synthesis, partial solid phasemethods, fragment condensation or classical solution synthesis. Thesesynthesis methods are well-known to those of skill in the art (see, forexample, Merrifield, J. Am. Chem. Soc. 85:2149 (1963), Stewart et al.,“Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co.1984), Bayer and Rapp, Chem. Pept. Prot. 3:3 (1986), Atherton et al.,Solid Phase Peptide Synthesis: A Practical Approach (IRL Press 1989),Fields and Colowick, “Solid-Phase Peptide Synthesis,” Methods inEnzymology Volume 289 (Academic Press 1997), and Lloyd-Williams et al.,Chemical Approaches to the Synthesis of Peptides and Proteins (CRCPress, Inc. 1997)). Variations in total chemical synthesis strategies,such as “native chemical ligation” and “expressed protein ligation” arealso standard (see, for example, Dawson et al., Science 266:776 (1994),Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997), Dawson,Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l Acad. Sci. USA95:6705 (1998), and Severinov and Muir, J. Biol. Chem. 273:16205(1998)).

5. Assays for TACI-Immunoglobulin Fusion Proteins

The function of TACI-immunoglobulin fusion proteins can be examinedusing a variety of approaches to assess the ability of the fusionproteins to bind ZTNF4 or ZTNF2. As an illustration, Example 4 providesmethods for measuring ZTNF4 binding affinity and binding capacity.

Alternatively, TACI-immunoglobulin fusion proteins can be characterizedby the ability to inhibit the stimulation of human B cells by solubleZTNF4, as described by Gross et al., international publication No.WO00/40716. Briefly, human B cells are isolated from peripheral bloodmononuclear cells using CD19 magnetic beads and the VarioMacs magneticseparation system (Miltenyi Biotec Auburn, Calif.) according to themanufacturer's instructions. Purified B cells are mixed with solubleZTNF4 (25 ng/ml) and recombinant human IL-4 (10 ng/ml Pharmingen), andthe cells are plated onto round bottom 96 well plates at 1×10⁵ cells perwell.

Soluble TACI-immunoglobulin proteins can be diluted from about 5 μg/mlto about 6 ng/ml, and incubated with the B cells for five days, pulsingovernight on day four with 1 μCi ³H-thymidine per well. As a control,TACI-immunoglobulin protein can also be incubated with B cells and IL-4without ZTNF4. Plates are harvested using Packard plate harvester, andcounted using the Packard reader.

This general approach was used to examine three TACI-Fc fusion proteins.Although all fusion proteins inhibited B cell proliferation, constructsTACI (d1-29, d111-154)-Fc5 and TACI (d1-29, d120-154)-Fc5 were morepotent than TACI (d1-29, d107-154)-Fc5.

Well-established animal models are available to test in vivo efficacy ofTACI-immunoglobulin proteins in certain disease states. For example,TACI-immunoglobulin proteins can be tested in a number of animal modelsof autoimmune disease, such as MRL-lpr/lpr or NZB×NZW F1 congenic mousestrains, which serve as a model of SLE (systemic lupus erythematosus).Such animal models are known in the art (see, for example, Cohen andMiller (Eds.), Autoimmune Disease Models: A Guidebook (Academic Press,Inc. 1994).

Offspring of a cross between New Zealand Black (NZB) and New ZealandWhite (NZW) mice develop a spontaneous form of SLE that closelyresembles SLE in humans. The offspring mice, known as NZBW begin todevelop IgM autoantibodies against T-cells at one month of age, and byfive to seven months of age, anti-DNA autoantibodies are the dominantimmunoglobulin. Polyclonal B-cell hyperactivity leads to overproductionof autoantibodies. The deposition of these autoantibodies, particularlythose directed against single stranded DNA, is associated with thedevelopment of glomerulonephritis, which manifests clinically asproteinuria, azotemia, and death from renal failure.

Kidney failure is the leading cause of death in mice affected withspontaneous SLE, and in the NZBW strain, this process is chronic andobliterative. The disease is more rapid and severe in females thanmales, with mean survival of only 245 days as compared to 406 days forthe males. While many of the female mice will be symptomatic(proteinuria) by seven to nine months of age, some can be much youngeror older when they develop symptoms. The fatal immune nephritis seen inthe NZBW mice is very similar to the glomerulonephritis seen in humanSLE, making this spontaneous murine model very attractive for testing ofpotential SLE therapeutics (Putterman and Naparstek, “Murine Models ofSpontaneous Systemic Lupus Erythematosus,” in Autoimmune Disease Models:A Guidebook, pages 217-234 (Academic Press, Inc., 1994); Mohan et al.,J. Immunol. 154:1470 (1995); and Daikh et al., J. Immunol. 159:3104(1997)).

As described by Gross et al., international publication No. WO00/40716,TACI-immunoglobulin proteins can be administered to NZBW mice to monitorits suppressive effect on B cells over the five-week period when, onaverage, B-cell autoantibody production is believed to be at high levelsin NZBW mice. Briefly, 100 8-week old female (NZB×NZW)F₁ mice can bedivided into six groups of 15 mice. Prior to treatment, the mice aremonitored once a month for urine protein, and blood is drawn for CBC andserum banking. Serum can be screened for the presence of autoantibodies.Because proteinuria is the hallmark sign of glomerulonephritis, urineprotein levels are monitored by dipstick at regular intervals over thecourse of the study. Treatment can begin when mice are approximatelyfive months of age. The mice receive intraperitoneal injections ofvehicle only (phosphate buffered saline) or human TACI-immunoglobulin(control protein) or TACI-immunoglobulin protein (e.g., 20 to 100 μgtest protein per dose) three times a week for five weeks.

Blood is collected twice during treatment, and will be collected atleast twice following treatment. Urine dipstick values for proteinuriaand body weights are determined every two weeks after treatment begins.Blood, urine dipstick value and body weight are collected at the time ofeuthanasia. The spleen and thymus are divided for fluorescent activatedcell sorting analysis and histology. Submandibular salivary glands,mesenteric lymph node chain, liver lobe with gall bladder, cecum andlarge intestine, stomach, small intestine, pancreas, right kidney,adrenal gland, tongue with trachea and esophagus, heart and lungs arealso collected for histology.

Murine models for experimental allergic encephalomyelitis have been usedas a tool to investigate both the mechanisms of immune-mediated disease,and methods of potential therapeutic intervention. The model resembleshuman multiple sclerosis, and produces demyelination as a result ofT-cell activation to neuroproteins such as myelin basic protein, orproteolipid protein. Inoculation with antigen leads to induction ofCD4+, class II MHC-restricted T-cells (Th1). Changes in the protocol forexperimental allergic encephalomyelitis can produce acute,chronic-relapsing, or passive-transfer variants of the model (Weinberget al., J. Immunol. 162:1818 (1999); Mijaba et al., Cell. Immunol.186:94 (1999); and Glabinski, Meth. Enzym. 288:182 (1997)).

Gross et al., international publication No. WO00/40716, describe oneapproach to evaluating the efficacy of TACI-immunoglobulin proteins inthe amelioration of symptoms associated with experimental allergicencephalomyelitis. Briefly, 25 female PL×SJL F1 mice (12 weeks old) aregiven a subcutaneous injection of 125 μg/mouse of antigen (myelinProteolipid Protein, PLP, residues 139-151), formulated in completeFreund's Adjuvant. The mice are divided into five groups of five mice.Intraperitoneal injections of pertussis toxin (400 ng) are given on Day0 and 2. The groups are given a 1×, 10×, or 100× dose ofTACI-immunoglobulin protein, one group will receive vehicle only, andone group will receive no treatment. Prevention therapy begins on Day 0,intervention therapy begins on day 7, or at onset of clinical signs.Signs of disease, weight loss, and paralysis manifest in approximately10 to 14 days, and last for about one week. Animals are assessed dailyby collecting body weights and assigning a clinical score to correspondto the extent of their symptoms. Clinical signs of experimental allergicencephalomyelitis appear within 10 to 14 days of inoculation and persistfor approximately one week. At the end of the study, all animals areeuthanized by gas overdose, and necropsied. The brain and spinal columnare collected for histology or frozen for mRNA analysis. Body weight andclinical score data are plotted by individual and by group.

In the collagen-induced arthritis model, mice develop chronicinflammatory arthritis, which closely resembles human rheumatoidarthritis. Since collagen-induced arthritis shares similar immunologicaland pathological features with rheumatoid arthritis, this makes it anideal model for screening potential human anti-inflammatory compounds.Another advantage in using the collagen-induced arthritis model is thatthe mechanisms of pathogenesis are known. The T and B cell epitopes ontype II collagen have been identified, and various immunological(delayed-type hypersensitivity and anti-collagen antibody) andinflammatory (cytokines, chemokines, and matrix-degrading enzymes)parameters relating to immune-mediating arthritis have been determined,and can be used to assess test compound efficacy in the models (Wooley,Curr. Opin. Rheum. 3:407 (1999); Williams et al., Immunol. 89:9784(1992); Myers et al., Life Sci. 61:1861 (1997); and Wang et al.,Immunol. 92:8955 (1995)).

Gross et al., international publication No. WO00/40716, describe amethod for evaluating the efficacy of TACI-immunoglobulin proteins inthe amelioration of symptoms associated with collagen-induced arthritis.In brief, eight-week old male DBA/1J mice (Jackson Labs) are dividedinto groups of five mice/group and are given two subcutaneous injectionsof 50 to 100 μl of 1 mg/ml collagen (chick or bovine origin), at threeweek intervals. One control does not receive collagen injections. Thefirst injection is formulated in Complete Freund's Adjuvant, and thesecond injection is formulated in Incomplete Freund's Adjuvant.TACI-immunoglobulin protein is administered prophylactically at orbefore the second injection, or after the animal develops a clinicalscore of two or more that persists at least 24 hours. Animals begin toshow symptoms of arthritis following the second collagen injection,usually within two to three weeks. For example, TACI-Fc, a controlprotein, human IgFc, or phosphate-buffered saline (vehicle) can beadministered prophylactically beginning seven days before the secondinjection (day—7). Proteins can be administered at 100 μg, given threetimes a week as a 200 μl intraperitoneal injection, and continued forfour weeks.

In the collagen-induced arthritis model, the extent of disease isevaluated in each paw using a caliper to measure paw thickness andassigning a clinical score to each paw. For example, a clinical score of“0” indicates a normal mouse, a score of “1” indicates that one or moretoes are inflamed, a score of “2” indicates mild paw inflammation, ascore of “3” indicates moderate paw inflammation, and a score of “4”indicates severe paw inflammation Animals are euthanized after thedisease as been established for a set period of time, usually sevendays. Paws are collected for histology or mRNA analysis, and serum iscollected for immunoglobulin and cytokine assays.

Myasthenia gravis is another autoimmune disease for which murine modelsare available. Myasthenia gravis is a disorder of neuromusculartransmission involving the production of autoantibodies directed againstthe nicotinic acetylcholine receptor. This disease is acquired orinherited with clinical features including abnormal weakness and fatigueon exertion.

A murine model of myasthenia gravis has been established. (Christadosset al., “Establishment of a Mouse Model of Myasthenia gravis WhichMimics Human Myasthenia gravid Pathogenesis for Immune Intervention,” inImmunobiology of Proteins and Peptides VIII, Atassi and Bixler (Eds.),pages 195-199 (1995)). Experimental autoimmune myasthenia gravis is anantibody mediated disease characterized by the presence of antibodies toacetylcholine receptor. These antibodies destroy the receptor leading todefective neuromuscular electrical impulses, resulting in muscleweakness. In the experimental autoimmune myasthenia gravis model, miceare immunized with the nicotinic acetylcholine receptor. Clinical signsof myasthenia gravis become evident weeks after the second immunization.Experimental autoimmune myasthenia gravis is evaluated by severalmethods including measuring serum levels of acetylcholine receptorantibodies by radioimmunoassay (Christadoss and Dauphinee, J. Immunol.136:2437 (1986); Lindstrom et al., Methods Enzymol. 74:432 (1981)),measuring muscle acetylcholine receptor, or electromyography (Coligan etal. (Eds.), Protocols in Immunology. Vol. 3, page 15.8.1 (John Wiley &Sons, 1997)).

The effect of TACI-immunoglobulin on experimental autoimmune myastheniagravis can be determined by administering fusion proteins during ongoingclinical myasthenia gravis in B6 mice. For example, 100 B6 mice areimmunized with 20 μg acetylcholine receptor in complete Freund'sadjuvant on days 0 and 30. Approximately 40 to 60% of mice will developmoderate (grade 2) to severe (grade 3) clinical myasthenia gravis afterthe boost with acetylcholine receptor. Mice with grade 2 and 3 clinicaldisease are divided into three groups (with equal grades of weakness)and weighed (mice with weakness also lose weight, since they havedifficulty in consuming food and water) and bled for serum (forpre-treatment anti-acetylcholine receptor antibody and isotype level).Group A is injected I.P with phosphate buffered saline, group B isinjected intraperitoneally with human IgG-Fc as a control protein (100μg), and group C is injected with 100 μg of TACI-Fc three times a weekfor four weeks. Mice are screened for clinical muscle weakness twice aweek, and weighed and bled for serum 15 and 30 days after thecommencement of treatment. Whole blood is collected on day 15 todetermine T/B cell ratio by fluorescence activated cell sorter analysisusing markers B220 and CD5. Surviving mice are killed 30 to 45 daysafter the initiation of treatment, and their carcasses are frozen forlater extraction of muscle acetylcholine receptor to determine the lossof muscle acetylcholine receptor, the primary pathology in myastheniagravis (see, for example, Coligan et al. (Eds.), Protocols inImmunology. Vol. 3, page 15.8.1 (John Wiley & Sons, 1997)).

Serum antibodies to mouse muscle acetylcholine receptor can bedetermined by an established radioimmunoassay, and anti-acetylcholinereceptor antibody isotypes (IgM, IgG1, IgG2b and IgG2c) is measured byELISA. Such methods are known. The effects of TACI-immunoglobulin onongoing clinical myasthenia gravis, anti-acetylcholine receptor antibodyand isotype level, and muscle acetylcholine receptor loss are determined

Approximately 100 mice can be immunized with 20 μg acetylcholinereceptor in complete Freund's adjuvant on day 0 and 30. Mice withclinical myasthenia gravis are divided into four groups. Group A isinjected intraperitoneally with 100 μg control Fc, group B is injectedwith 20 μg control Fc, group C is injected with 100 μg TACI-Fc, andgroup D is injected with 20 μg TACI-Fc three times a week for fourweeks. Mice are weighed and bled for serum before, and 15 and 30 daysafter the start of the treatment. Serum is tested for anti-acetylcholinereceptor antibody and isotypes as described above. Muscle acetylcholinereceptor loss can also be measured.

Other suitable assays of TACI-immunoglobulin fusion proteins can bedetermined by those of skill in the art.

6. Production of TACI-Immunoglobulin Conjugates

The present invention includes chemically modified TACI-immunoglobulincompositions, in which a TACI-immunoglobulin polypeptide is linked witha polymer. Typically, the polymer is water-soluble so that theTACI-immunoglobulin conjugate does not precipitate in an aqueousenvironment, such as a physiological environment. An example of asuitable polymer is one that has been modified to have a single reactivegroup, such as an active ester for acylation, or an aldehyde foralkylation, In this way, the degree of polymerization can be controlled.An example of a reactive aldehyde is polyethylene glycolpropionaldehyde, or mono-(C₁-C₁₀)alkoxy, or aryloxy derivatives thereof(see, for example, Harris, et al., U.S. Pat. No. 5,252,714). The polymermay be branched or unbranched. Moreover, a mixture of polymers can beused to produce TACI-immunoglobulin conjugates.

TACI-immunoglobulin conjugates used for therapy can comprisepharmaceutically acceptable water-soluble polymer moieties. Suitablewater-soluble polymers include polyethylene glycol (PEG),monomethoxy-PEG, mono-(C₁-C₁₀)alkoxy-PEG, aryloxy-PEG, poly-(N-vinylpyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde,bis-succinimidyl carbonate PEG, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, dextran, cellulose, or othercarbohydrate-based polymers. Suitable PEG may have a molecular weightfrom about 600 to about 60,000, including, for example, 5,000, 12,000,20,000 and 25,000. A TACI-immunoglobulin conjugate can also comprise amixture of such water-soluble polymers.

One example of a TACI-immunoglobulin conjugate comprises aTACI-immunoglobulin moiety and a polyalkyl oxide moiety attached to theN-terminus of the TACI-immunoglobulin. PEG is one suitable polyalkyloxide. As an illustration, TACI-immunoglobulin can be modified with PEG,a process known as “PEGylation.” PEGylation of TACI-immunoglobulin canbe carried out by any of the PEGylation reactions known in the art (see,for example, EP 0 154 316, Delgado et al., Critical Reviews inTherapeutic Drug Carrier Systems 9:249 (1992), Duncan and Spreafico,Clin. Pharmacokinet. 27:290 (1994), and Francis et al., Int J Hematol68:1 (1998)). For example, PEGylation can be performed by an acylationreaction or by an alkylation reaction with a reactive polyethyleneglycol molecule. In an alternative approach, TACI-immunoglobulinconjugates are formed by condensing activated PEG, in which a terminalhydroxy or amino group of PEG has been replaced by an activated linker(see, for example, Karasiewicz et al., U.S. Pat. No. 5,382,657).

PEGylation by acylation typically requires reacting an active esterderivative of PEG with a TACI-immunoglobulin polypeptide. An example ofan activated PEG ester is PEG esterified to N-hydroxysuccinimide. Asused herein, the term “acylation” includes the following types oflinkages between TACI-immunoglobulin and a water-soluble polymer: amide,carbamate, urethane, and the like. Methods for preparing PEGylatedTACI-immunoglobulin by acylation will typically comprise the steps of(a) reacting a TACI-immunoglobulin polypeptide with PEG (such as areactive ester of an aldehyde derivative of PEG) under conditionswhereby one or more PEG groups attach to TACI-immunoglobulin, and (b)obtaining the reaction product(s). Generally, the optimal reactionconditions for acylation reactions will be determined based upon knownparameters and desired results. For example, the larger the ratio ofPEG:TACI-immunoglobulin, the greater the percentage of polyPEGylatedTACI-immunoglobulin product.

The product of PEGylation by acylation is typically a polyPEGylatedTACI-immunoglobulin product, wherein the lysine ε-amino groups arePEGylated via an acyl linking group. An example of a connecting linkageis an amide. Typically, the resulting TACI-immunoglobulin will be atleast 95% mono-, di-, or tri-pegylated, although some species withhigher degrees of PEGylation may be formed depending upon the reactionconditions. PEGylated species can be separated from unconjugatedTACI-immunoglobulin polypeptides using standard purification methods,such as dialysis, ultrafiltration, ion exchange chromatography, affinitychromatography, and the like.

PEGylation by alkylation generally involves reacting a terminal aldehydederivative of PEG with TACI-immunoglobulin in the presence of a reducingagent. PEG groups can be attached to the polypeptide via a —CH₂—NHgroup.

Derivatization via reductive alkylation to produce a monoPEGylatedproduct takes advantage of the differential reactivity of differenttypes of primary amino groups available for derivatization. Typically,the reaction is performed at a pH that allows one to take advantage ofthe pKa differences between the ε-amino groups of the lysine residuesand the α-amino group of the N-terminal residue of the protein. By suchselective derivatization, attachment of a water-soluble polymer thatcontains a reactive group such as an aldehyde, to a protein iscontrolled. The conjugation with the polymer occurs predominantly at theN-terminus of the protein without significant modification of otherreactive groups such as the lysine side chain amino groups. The presentinvention provides a substantially homogenous preparation ofTACI-immunoglobulin monopolymer conjugates.

Reductive alkylation to produce a substantially homogenous population ofmonopolymer TACI-immunoglobulin conjugate molecule can comprise thesteps of: (a) reacting a TACI-immunoglobulin polypeptide with a reactivePEG under reductive alkylation conditions at a pH suitable to permitselective modification of the α-amino group at the amino terminus of theTACI-immunoglobulin, and (b) obtaining the reaction product(s). Thereducing agent used for reductive alkylation should be stable in aqueoussolution and able to reduce only the Schiff base formed in the initialprocess of reductive alkylation. Illustrative reducing agents includesodium borohydride, sodium cyanoborohydride, dimethylamine borane,trimethylamine borane, and pyridine borane.

For a substantially homogenous population of monopolymerTACI-immunoglobulin conjugates, the reductive alkylation reactionconditions are those that permit the selective attachment of the watersoluble polymer moiety to the N-terminus of TACI-immunoglobulin. Suchreaction conditions generally provide for pKa differences between thelysine amino groups and the α-amino group at the N-terminus. The pH alsoaffects the ratio of polymer to protein to be used. In general, if thepH is lower, a larger excess of polymer to protein will be desiredbecause the less reactive the N-terminal α-group, the more polymer isneeded to achieve optimal conditions. If the pH is higher, thepolymer:TACI-immunoglobulin need not be as large because more reactivegroups are available. Typically, the pH will fall within the range of 3to 9, or 3 to 6.

Another factor to consider is the molecular weight of the water-solublepolymer. Generally, the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.For PEGylation reactions, the typical molecular weight is about 2 kDa toabout 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25kDa. The molar ratio of water-soluble polymer to TACI-immunoglobulinwill generally be in the range of 1:1 to 100:1. Typically, the molarratio of water-soluble polymer to TACI-immunoglobulin will be 1:1 to20:1 for polyPEGylation, and 1:1 to 5:1 for monoPEGylation.

General methods for producing conjugates comprising a polypeptide andwater-soluble polymer moieties are known in the art. See, for example,Karasiewicz et al., U.S. Pat. No. 5,382,657, Greenwald et al., U.S. Pat.No. 5,738,846, Nieforth et al., Clin. Pharmacol. Ther. 59:636 (1996),Monkarsh et al., Anal. Biochem. 247:434 (1997)).

The present invention contemplates compositions comprising a peptide orpolypeptide described herein. Such compositions can further comprise acarrier. The carrier can be a conventional organic or inorganic carrier.Examples of carriers include water, buffer solution, alcohol, propyleneglycol, macrogol, sesame oil, corn oil, and the like.

7. Isolation of TACI-Immunoglobulin Polypeptides

The polypeptides of the present invention can be purified to at leastabout 80% purity, to at least about 90% purity, to at least about 95%purity, or greater than 95% purity with respect to contaminatingmacromolecules, particularly other proteins and nucleic acids, and freeof infectious and pyrogenic agents. The polypeptides of the presentinvention may also be purified to a pharmaceutically pure state, whichis greater than 99.9% pure. In certain preparations, purifiedpolypeptide is substantially free of other polypeptides, particularlyother polypeptides of animal origin.

Fractionation and/or conventional purification methods can be used toobtain preparations of synthetic TACI-immunoglobulin polypeptides, andrecombinant TACI-immunoglobulin polypeptides purified from recombinanthost cells. In general, ammonium sulfate precipitation and acid orchaotrope extraction may be used for fractionation of samples. Exemplarypurification steps may include hydroxyapatite, size exclusion, FPLC andreverse-phase high performance liquid chromatography. Suitablechromatographic media include derivatized dextrans, agarose, cellulose,polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Qderivatives are suitable. Exemplary chromatographic media include thosemedia derivatized with phenyl, butyl, or octyl groups, such asPhenyl-Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas,Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia) and the like; orpolyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.Suitable solid supports include glass beads, silica-based resins,cellulosic resins, agarose beads, cross-linked agarose beads,polystyrene beads, cross-linked polyacrylamide resins and the like thatare insoluble under the conditions in which they are to be used. Thesesupports may be modified with reactive groups that allow attachment ofproteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxylgroups and/or carbohydrate moieties.

Examples of coupling chemistries include cyanogen bromide activation,N-hydroxysuccinimide activation, epoxide activation, sulfhydrylactivation, hydrazide activation, and carboxyl and amino derivatives forcarbodiimide coupling chemistries. These and other solid media are wellknown and widely used in the art, and are available from commercialsuppliers. Selection of a particular method for polypeptide isolationand purification is a matter of routine design and is determined in partby the properties of the chosen support. See, for example, AffinityChromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988),and Doonan, Protein Purification Protocols (The Humana Press 1996).

Additional variations in TACI-immunoglobulin isolation and purificationcan be devised by those of skill in the art. For example, anti-TACI oranti-Fc antibodies can be used to isolate large quantities of protein byimmunoaffinity purification.

The polypeptides of the present invention can also be isolated byexploitation of particular properties. For example, immobilized metalion adsorption (IMAC) chromatography can be used to purifyhistidine-rich proteins, including those comprising polyhistidine tags.Briefly, a gel is first charged with divalent metal ions to form achelate (Sulkowski, Trends in Biochem. 3:1 (1985)). Histidine-richproteins will be adsorbed to this matrix with differing affinities,depending upon the metal ion used, and will be eluted by competitiveelution, lowering the pH, or use of strong chelating agents. Othermethods of purification include purification of glycosylated proteins bylectin affinity chromatography, Protein A chromatography, and ionexchange chromatography (M. Deutscher, (ed.), Meth. Enzymol. 182:529(1990)).

TACI-immunoglobulin polypeptides or fragments thereof may also beprepared through chemical synthesis, as described above.TACI-immunoglobulin polypeptides may be monomers or multimers;glycosylated or non-glycosylated; PEGylated or non-PEGylated; and may ormay not include an initial methionine amino acid residue. ATACI-immunoglobulin fusion protein may be non-glycosylated,glycosylated, or glycosylated only in the TACI moiety or in theimmunoglobulin moiety. The immunoglobulin moiety can be obtained from ahuman antibody, a chimeric antibody, or a humanized antibody.

8. Therapeutic Uses of TACI-Immunoglobulin Polypeptides

TACI-immunoglobulin proteins can be used to modulate the immune systemby binding ZTNF4 or ZTNF2, and thus, preventing the binding of theseligands with endogenous TACI or BCMA receptors. Accordingly, the presentinvention includes the use of TACI-immunoglobulin proteins to a subject,which lacks an adequate amount of TACI or BCMA receptors, or whichproduces an excess of ZTNF4 or ZTNF2. These molecules can beadministered to any subject in need of treatment, and the presentinvention contemplates both veterinary and human therapeutic uses.Illustrative subjects include mammalian subjects, such as farm animals,domestic animals, and human patients.

TACI-immunoglobulin polypeptides can be used for the treatment ofautoimmune diseases, B cell cancers, immunomodulation, IBD and anyantibody-mediated pathologies (e.g., ITCP, myasthenia gravis and thelike), renal diseases, indirect T cell immune response, graft rejection,and graft versus host disease. The polypeptides of the present inventioncan be targeted to specifically regulate B cell responses during theimmune response. Additionally, the polypeptides of the present inventioncan be used to modulate B cell development, development of other cells,antibody production, and cytokine production. Polypeptides of thepresent invention can also modulate T and B cell communication byneutralizing the proliferative effects of ZTNF4.

TACI-immunoglobulin polypeptides of the present invention can be usefulto neutralize the effects of ZTNF4 for treating pre-B or B-cellleukemias, such as plasma cell leukemia, chronic or acute lymphocyticleukemia, myelomas such as multiple myeloma, plasma cell myeloma,endothelial myeloma and giant cell myeloma, and lymphomas such asnon-Hodgkins lymphoma, for which an increase in ZTNF4 polypeptides isassociated.

ZTNF4 is expressed in CD8⁺ cells, monocytes, dendritic cells, activatedmonocytes, which indicates that, in certain autoimmune disorders,cytotoxic T-cells might stimulate B-cell production through excessproduction of ZTNF4. Immunosuppressant proteins that selectively blockthe action of B-lymphocytes would be of use in treating disease.Autoantibody production is common to several autoimmune diseases andcontributes to tissue destruction and exacerbation of disease.Autoantibodies can also lead to the occurrence of immune complexdeposition complications and lead to many symptoms of systemic lupuserythematosus, including kidney failure, neuralgic symptoms and death.Modulating antibody production independent of cellular response wouldalso be beneficial in many disease states. B cells have also been shownto play a role in the secretion of arthritogenic immunoglobulins inrheumatoid arthritis. As such, inhibition of ZTNF4 antibody productionwould be beneficial in treatment of autoimmune diseases such asmyasthenia gravis, rheumatoid arthritis, polyarticular-course juvenilerheumatoid arthritis, and psoriatic arthritis Immunosuppressanttherapeutics such as TACI-immunoglobulin proteins that selectively blockor neutralize the action of B-lymphocytes would be useful for suchpurposes.

The invention provides methods employing TACI-immunoglobulin proteinsfor selectively blocking or neutralizing the actions of B-cells inassociation with end stage renal diseases, which may or may not beassociated with autoimmune diseases. Such methods would also be usefulfor treating immunologic renal diseases. Such methods would be would beuseful for treating glomerulonephritis associated with diseases such asmembranous nephropathy, IgA nephropathy or Berger's Disease, IgMnephropathy, Goodpasture's Disease, post-infectious glomerulonephritis,mesangioproliferative disease, chronic lymphoid leukemia, minimal-changenephrotic syndrome. Such methods would also serve as therapeuticapplications for treating secondary glomerulonephritis or vasculitisassociated with such diseases as lupus, polyarteritis, Henoch-Schonlein,Scleroderma, HIV-related diseases, amyloidosis or hemolytic uremicsyndrome. The methods of the present invention would also be useful aspart of a therapeutic application for treating interstitial nephritis orpyelonephritis associated with chronic pyelonephritis, analgesic abuse,nephrocalcinosis, nephropathy caused by other agents, nephrolithiasis,or chronic or acute interstitial nephritis.

The methods of the present invention also include use ofTACI-immunoglobulin proteins in the treatment of hypertensive or largevessel diseases, including renal artery stenosis or occlusion andcholesterol emboli or renal emboli.

The present invention also provides methods for treatment of renal orurological neoplasms, multiple myelomas, lymphomas, light chainneuropathy or amyloidosis.

The invention also provides methods for blocking or inhibiting activatedB cells using TACI-immunoglobulin proteins for the treatment of asthmaand other chronic airway diseases such as bronchitis and emphysema. TheTACI-immunoglobulin proteins described herein can also be used to treatSjögren's Syndrome.

Also provided are methods for inhibiting or neutralizing an effector Tcell response using TACI-immunoglobulin proteins for use inimmunosuppression, in particular for such therapeutic use as forgraft-versus-host disease and graft rejection. Moreover,TACI-immunoglobulin proteins would be useful in therapeutic protocolsfor treatment of such autoimmune diseases as insulin dependent diabetesmellitus (IDDM) and Crohn's Disease. Methods of the present inventionwould have additional therapeutic value for treating chronicinflammatory diseases, in particular to lessen joint pain, swelling,anemia and other associated symptoms as well as treating septic shock.

Well established animal models are available to test in vivo efficacy ofTACI-immunoglobulin proteins of the present invention in certain diseasestates. In particular, TACI-immunoglobulin proteins can be tested invivo in a number of animal models of autoimmune disease, such asMRL-lpr/lpr or NZB×NZW F1 congenic mouse strains which serve as a modelof SLE (systemic lupus erythematosus). Such animal models are known inthe art.

Offspring of a cross between New Zealand Black (NZB) and New ZealandWhite (NZW) mice develop a spontaneous form of SLE that closelyresembles SLE in humans. The offspring mice, known as NZBW begin todevelop IgM autoantibodies against T-cells at 1 month of age, and by 5-7months of age, Ig anti-DNA autoantibodies are the dominantimmunoglobulin. Polyclonal B-cell hyperactivity leads to overproductionof autoantibodies. The deposition of these autoantibodies, particularlyones directed against single stranded DNA is associated with thedevelopment of glomerulonephritis, which manifests clinically asproteinuria, azotemia, and death from renal failure. Kidney failure isthe leading cause of death in mice affected with spontaneous SLE, and inthe NZBW strain, this process is chronic and obliterative. The diseaseis more rapid and severe in females than males, with mean survival ofonly 245 days as compared to 406 days for the males. While many of thefemale mice will be symptomatic (proteinuria) by 7-9 months of age, somecan be much younger or older when they develop symptoms. The fatalimmune nephritis seen in the NZBW mice is very similar to theglomerulonephritis seen in human SLE, making this spontaneous murinemodel useful for testing of potential SLE therapeutics.

Mouse models for experimental allergic encephalomyelitis (EAE) has beenused as a tool to investigate both the mechanisms of immune-mediateddisease, and methods of potential therapeutic intervention. The modelresembles human multiple sclerosis, and produces demyelination as aresult of T-cell activation to neuroproteins such as myelin basicprotein (MBP), or proteolipid protein (PLP). Inoculation with antigenleads to induction of CD4+, class II MHC-restricted T-cells (Th1).Changes in the protocol for EAE can produce acute, chronic-relapsing, orpassive-transfer variants of the model.

In the collagen-induced arthritis (CIA) model, mice develop chronicinflammatory arthritis, which closely resembles human rheumatoidarthritis (RA). Since CIA shares similar immunological and pathologicalfeatures with RA, this makes it an ideal model for screening potentialhuman anti-inflammatory compounds. Another advantage in using the CIAmodel is that the mechanisms of pathogenesis are known. The T and B cellepitopes on type II collagen have been identified, and variousimmunological (delayed-type hypersensitivity and anti-collagen antibody)and inflammatory (cytokines, chemokines, and matrix-degrading enzymes)parameters relating to immune-mediating arthritis have been determined,and can be used to assess test compound efficacy in the models.

Myasthenia gravis (MG) is another autoimmune disease for which murinemodels are available. MG is a disorder of neuromuscular transmissioninvolving the production of autoantibodies directed against thenicotinic acetylcholine receptor (AChR). MG is acquired or inheritedwith clinical features including abnormal weakness and fatigue onexertion. A mouse model of MG has been established. Experimentalautoimmune myasthenia gravis (EAMG) is an antibody mediated diseasecharacterized by the presence of antibodies to AChR. These antibodiesdestroy the receptor leading to defective neuromuscular electricalimpulses, resulting in muscle weakness. In the EAMG model, mice areimmunized with the nicotinic acetylcholine receptor. Clinical signs ofMG become evident weeks after the second immunization. EAMG is evaluatedby several methods including measuring serum levels of AChR antibodiesby radioimmunoassay, measuring muscle AChR, or electromyography.

Generally, the dosage of administered TACI-immunoglobulin protein willvary depending upon such factors as the subject's age, weight, height,sex, general medical condition and previous medical history. Typically,it is desirable to provide the recipient with a dosage ofTACI-immunoglobulin protein, which is in the range of from about 1 pg/kgto 10 mg/kg (amount of agent/body weight of subject), although a loweror higher dosage also may be administered as circumstances dictate.

Administration of a TACI-immunoglobulin protein to a subject can beintravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, intrapleural, intrathecal, by perfusion through a regionalcatheter, or by direct intralesional injection. When administeringtherapeutic proteins by injection, the administration may be bycontinuous infusion or by single or multiple boluses.

Additional routes of administration include oral, mucosal-membrane,pulmonary, and transcutaneous. Oral delivery is suitable for polyestermicrospheres, zein microspheres, proteinoid microspheres,polycyanoacrylate microspheres, and lipid-based systems (see, forexample, DiBase and Morrel, “Oral Delivery of MicroencapsulatedProteins,” in Protein Delivery: Physical Systems, Sanders and Hendren(eds.), pages 255-288 (Plenum Press 1997)). The feasibility of anintranasal delivery is exemplified by such a mode of insulinadministration (see, for example, Hinchcliffe and Illum, Adv. DrugDeliv. Rev. 35:199 (1999)). Dry or liquid particles comprisingTACI-immunoglobulin can be prepared and inhaled with the aid ofdry-powder dispersers, liquid aerosol generators, or nebulizers (e.g.,Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al., Adv. DrugDeliv. Rev. 35:235 (1999)). This approach is illustrated by the AERXdiabetes management system, which is a hand-held electronic inhaler thatdelivers aerosolized insulin into the lungs. Studies have shown thatproteins as large as 48,000 kDa have been delivered across skin attherapeutic concentrations with the aid of low-frequency ultrasound,which illustrates the feasibility of transcutaneous administration(Mitragotri et al., Science 269:850 (1995)). Transdermal delivery usingelectroporation provides another means to administer aTACI-immunoglobulin protein (Potts et al., Pharm. Biotechnol. 10:213(1997)).

A pharmaceutical composition comprising a TACI-immunoglobulin proteincan be formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby the therapeutic proteins are combined in amixture with a pharmaceutically acceptable carrier. A composition issaid to be a “pharmaceutically acceptable carrier” if its administrationcan be tolerated by a recipient patient. Sterile phosphate-bufferedsaline is one example of a pharmaceutically acceptable carrier. Othersuitable carriers are well-known to those in the art. See, for example,Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (MackPublishing Company 1995).

For purposes of therapy, TACI-immunoglobulin proteins are administeredto a patient in a therapeutically effective amount. ATACI-immunoglobulin protein and a pharmaceutically acceptable carrier issaid to be administered in a “therapeutically effective amount” if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient. For example, an agentused to treat inflammation is physiologically significant if itspresence alleviates the inflammatory response. As another example, anagent used to inhibit the growth of tumor cells is physiologicallysignificant if the administration of the agent results in a decrease inthe number of tumor cells, decreased metastasis, a decrease in the sizeof a solid tumor, or increased necrosis of a tumor. Furthermore, anagent used to treat systemic lupus erythematosus is physiologicallysignificant if the administration of the agent results in a decrease ofcirculating anti-double stranded DNA antibodies, or a decrease in atleast one of the following symptoms: fever, joint pain, erythematosusskin lesions, or other features of systemic lupus erythematosus. Oneexample of a general indication that a TACI-immunoglobulin protein isadministered in a therapeutically effective amount is that, followingadministration to a subject, there is a decrease in circulating levelsof ZTNF4 (BLyS).

A pharmaceutical composition comprising a TACI-immunoglobulin proteincan be furnished in liquid form, in an aerosol, or in solid form. Liquidforms, are illustrated by injectable solutions and oral suspensions.Exemplary solid forms include capsules, tablets, and controlled-releaseforms. The latter form is illustrated by miniosmotic pumps and implants(Bremer et al., Pharm. Biotechnol. 10:239 (1997); Ranade, “Implants inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 95-123 (CRC Press 1995); Bremer et al., “Protein Delivery withInfusion Pumps,” in Protein Delivery: Physical Systems, Sanders andHendren (eds.), pages 239-254 (Plenum Press 1997); Yewey et al.,“Delivery of Proteins from a Controlled Release Injectable Implant,” inProtein Delivery: Physical Systems, Sanders and Hendren (eds.), pages93-117 (Plenum Press 1997)).

Liposomes provide one means to deliver therapeutic polypeptides to asubject intravenously, intraperitoneally, intrathecally,intramuscularly, subcutaneously, or via oral administration, inhalation,or intranasal administration. Liposomes are microscopic vesicles thatconsist of one or more lipid bilayers surrounding aqueous compartments(see, generally, Bakker-Woudenberg et al., Eur. J. Clin. Microbiol.Infect. Dis. 12 (Suppl. 1):S61 (1993), Kim, Drugs 46:618 (1993), andRanade, “Site-Specific Drug Delivery Using Liposomes as Carriers,” inDrug Delivery Systems, Ranade and Hollinger (eds.), pages 3-24 (CRCPress 1995)). Liposomes are similar in composition to cellular membranesand as a result, liposomes can be administered safely and arebiodegradable. Depending on the method of preparation, liposomes may beunilamellar or multilamellar, and liposomes can vary in size withdiameters ranging from 0.02 μm to greater than 10 μm. A variety ofagents can be encapsulated in liposomes: hydrophobic agents partition inthe bilayers and hydrophilic agents partition within the inner aqueousspace(s) (see, for example, Machy et al., Liposomes In Cell Biology AndPharmacology (John Libbey 1987), and Ostro et al., American J. Hosp.Pharm. 46:1576 (1989)). Moreover, it is possible to control thetherapeutic availability of the encapsulated agent by varying liposomesize, the number of bilayers, lipid composition, as well as the chargeand surface characteristics of the liposomes.

Liposomes can adsorb to virtually any type of cell and then slowlyrelease the encapsulated agent. Alternatively, an absorbed liposome maybe endocytosed by cells that are phagocytic. Endocytosis is followed byintralysosomal degradation of liposomal lipids and release of theencapsulated agents (Scherphof et al., Ann. N.Y. Acad. Sci. 446:368(1985)). After intravenous administration, small liposomes (0.1 to 1.0μm) are typically taken up by cells of the reticuloendothelial system,located principally in the liver and spleen, whereas liposomes largerthan 3.0 μm are deposited in the lung. This preferential uptake ofsmaller liposomes by the cells of the reticuloendothelial system hasbeen used to deliver chemotherapeutic agents to macrophages and totumors of the liver.

The reticuloendothelial system can be circumvented by several methodsincluding saturation with large doses of liposome particles, orselective macrophage inactivation by pharmacological means (Claassen etal., Biochim. Biophys. Acta 802:428 (1984)). In addition, incorporationof glycolipid- or polyethelene glycol-derivatized phospholipids intoliposome membranes has been shown to result in a significantly reduceduptake by the reticuloendothelial system (Allen et al., Biochim.Biophys. Acta 1068:133 (1991); Allen et al., Biochim. Biophys. Acta1150:9 (1993)).

Liposomes can also be prepared to target particular cells or organs byvarying phospholipid composition or by inserting receptors or ligandsinto the liposomes. For example, liposomes, prepared with a high contentof a nonionic surfactant, have been used to target the liver (Hayakawaet al., Japanese Patent 04-244,018; Kato et al., Biol. Pharm. Bull.16:960 (1993)). These formulations were prepared by mixing soybeanphospatidylcholine, α-tocopherol, and ethoxylated hydrogenated castoroil (HCO-60) in methanol, concentrating the mixture under vacuum, andthen reconstituting the mixture with water. A liposomal formulation ofdipalmitoylphosphatidylcholine (DPPC) with a soybean-derivedsterylglucoside mixture (SG) and cholesterol (Ch) has also been shown totarget the liver (Shimizu et al., Biol. Pharm. Bull. 20:881 (1997)).

Alternatively, various targeting ligands can be bound to the surface ofthe liposome, such as antibodies, antibody fragments, carbohydrates,vitamins, and transport proteins. For example, liposomes can be modifiedwith branched type galactosyllipid derivatives to targetasialoglycoprotein (galactose) receptors, which are exclusivelyexpressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev.Ther. Drug Carrier Syst. 14:287 (1997); Murahashi et al., Biol. Pharm.Bull. 20:259 (1997)). Similarly, Wu et al., Hepatology 27:772 (1998),have shown that labeling liposomes with asialofetuin led to a shortenedliposome plasma half-life and greatly enhanced uptake ofasialofetuin-labeled liposome by hepatocytes. On the other hand, hepaticaccumulation of liposomes comprising branched type galactosyllipidderivatives can be inhibited by preinjection of asialofetuin (Murahashiet al., Biol. Pharm. Bull.20:259 (1997)). Polyaconitylated human serumalbumin liposomes provide another approach for targeting liposomes toliver cells (Kamps et al., Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).Moreover, Geho, et al. U.S. Pat. No. 4,603,044, describe ahepatocyte-directed liposome vesicle delivery system, which hasspecificity for hepatobiliary receptors associated with the specializedmetabolic cells of the liver.

In a more general approach to tissue targeting, target cells areprelabeled with biotinylated antibodies specific for a ligand expressedby the target cell (Harasym et al., Adv. Drug Deliv. Rev. 32:99 (1998)).After plasma elimination of free antibody, streptavidin-conjugatedliposomes are administered. In another approach, targeting antibodiesare directly attached to liposomes (Harasym et al., Adv. Drug Deliv.Rev. 32:99 (1998)).

TACI-immunoglobulin proteins can be encapsulated within liposomes usingstandard techniques of protein microencapsulation (see, for example,Anderson et al., Infect. Immun. 31:1099 (1981), Anderson et al., CancerRes. 50:1853 (1990), and Cohen et al., Biochim. Biophys. Acta 1063:95(1991), Alving et al. “Preparation and Use of Liposomes in ImmunologicalStudies,” in Liposome Technology, 2nd Edition, Vol. III, Gregoriadis(ed.), page 317 (CRC Press 1993), Wassef et al., Meth. Enzymol. 149:124(1987)). As noted above, therapeutically useful liposomes may contain avariety of components. For example, liposomes may comprise lipidderivatives of poly(ethylene glycol) (Allen et al., Biochim. Biophys.Acta 1150:9 (1993)).

Degradable polymer microspheres have been designed to maintain highsystemic levels of therapeutic proteins. Microspheres are prepared fromdegradable polymers such as poly(lactide-co-glycolide) (PLG),polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetatepolymers, in which proteins are entrapped in the polymer (Gombotz andPettit, Bioconjugate Chem. 6:332 (1995); Ranade, “Role of Polymers inDrug Delivery,” in Drug Delivery Systems, Ranade and Hollinger (eds.),pages 51-93 (CRC Press 1995); Roskos and Maskiewicz, “DegradableControlled Release Systems Useful for Protein Delivery,” in ProteinDelivery: Physical Systems, Sanders and Hendren (eds.), pages 45-92(Plenum Press 1997); Bartus et al., Science 281:1161 (1998); Putney andBurke, Nature Biotechnology 16:153 (1998); Putney, Curr. Opin. Chem.Biol. 2:548 (1998)). Polyethylene glycol (PEG)-coated nanospheres canalso provide carriers for intravenous administration of therapeuticproteins (see, for example, Gref et al., Pharm. Biotechnol. 10:167(1997)).

The present invention also contemplates chemically modifiedTACI-immunoglobulin proteins in which the polypeptide is linked with apolymer, as discussed above.

Other dosage forms can be devised by those skilled in the art, as shown,for example, by Ansel and Popovich, Pharmaceutical Dosage Forms and DrugDelivery Systems, 5^(th) Edition (Lea & Febiger 1990), Gennaro (ed.),Remington's Pharmaceutical Sciences, 19^(th) Edition (Mack PublishingCompany 1995), and by Ranade and Hollinger, Drug Delivery Systems (CRCPress 1996).

As an illustration, pharmaceutical compositions may be supplied as a kitcomprising a container that comprises a TACI-immunoglobulin protein.Therapeutic polypeptides can be provided in the form of an injectablesolution for single or multiple doses, or as a sterile powder that willbe reconstituted before injection. Alternatively, such a kit can includea dry-powder disperser, liquid aerosol generator, or nebulizer foradministration of a therapeutic polypeptide. Such a kit may furthercomprise written information on indications and usage of thepharmaceutical composition. Moreover, such information may include astatement that the TACI-immunoglobulin protein composition iscontraindicated in patients with known hypersensitivity to either theTACI receptor moiety or the immunoglobulin moiety.

9. Therapeutic Uses of TACI-Immunoglobulin Nucleotide Sequences

The present invention includes the use of nucleic acid molecules thatencode TACI-immunoglobulin fusion proteins to provide these fusionproteins to a subject in need of such treatment. For veterinarytherapeutic use or human therapeutic use, such nucleic acid moleculescan be administered to a subject having a disorder or disease, asdiscussed above. As one example discussed earlier, nucleic acidmolecules encoding a TACI-immunoglobulin fusion protein can be used forlong-term treatment of systemic lupus erythematosus.

There are numerous approaches for introducing a TACI-immunoglobulin geneto a subject, including the use of recombinant host cells that expressTACI-immunoglobulin, delivery of naked nucleic acid encodingTACI-immunoglobulin, use of a cationic lipid carrier with a nucleic acidmolecule that encodes TACI-immunoglobulin, and the use of viruses thatexpress TACI-immunoglobulin, such as recombinant retroviruses,recombinant adeno-associated viruses, recombinant adenoviruses, andrecombinant Herpes simplex viruses (see, for example, Mulligan, Science260:926 (1993), Rosenberg et al., Science 242:1575 (1988), LaSalle etal., Science 259:988 (1993), Wolff et al., Science 247:1465 (1990),Breakfield and Deluca, The New Biologist 3:203 (1991)). In an ex vivoapproach, for example, cells are isolated from a subject, transfectedwith a vector that expresses a TACI-immunoglobulin gene, and thentransplanted into the subject.

In order to effect expression of a TACI-immunoglobulin gene, anexpression vector is constructed in which a nucleotide sequence encodinga TACI-immunoglobulin gene is operably linked to a core promoter, andoptionally a regulatory element, to control gene transcription. Thegeneral requirements of an expression vector are described above.

Alternatively, a TACI-immunoglobulin gene can be delivered usingrecombinant viral vectors, including for example, adenoviral vectors(e.g., Kass-Eisler et al., Proc. Nat'l Acad. Sci. USA 90:11498 (1993),Kolls et al., Proc. Nat'l Acad. Sci. USA 91:215 (1994), Li et al., Hum.Gene Ther. 4:403 (1993), Vincent et al., Nat. Genet. 5:130 (1993), andZabner et al., Cell 75:207 (1993)), adenovirus-associated viral vectors(Flotte et al., Proc. Nat'l Acad. Sci. USA 90:10613 (1993)),alphaviruses such as Semliki Forest Virus and Sindbis Virus (Hertz andHuang, J. Vir. 66:857 (1992), Raju and Huang, J. Vir. 65:2501 (1991),and Xiong et al., Science 243:1188 (1989)), herpes viral vectors (e.g.,U.S. Pat. Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688),parvovirus vectors (Koering et al., Hum. Gene Therap. 5:457 (1994)), poxvirus vectors (Ozaki et al., Biochem. Biophys. Res. Comm. 193:653(1993), Panicali and Paoletti, Proc. Nat'l Acad. Sci. USA 79:4927(1982)), pox viruses, such as canary pox virus or vaccinia virus(Fisher-Hoch et al., Proc. Nat'l Acad. Sci. USA 86:317 (1989), andFlexner et al., Ann. N.Y. Acad. Sci. 569:86 (1989)), and retroviruses(e.g., Baba et al., J. Neurosurg 79:729 (1993), Ram et al., Cancer Res.53:83 (1993), Takamiya et al., J. Neurosci. Res 33:493 (1992), Vile andHart, Cancer Res. 53:962 (1993), Vile and Hart, Cancer Res. 53:3860(1993), and Anderson et al., U.S. Pat. No. 5,399,346). Within variousembodiments, either the viral vector itself, or a viral particle, whichcontains the viral vector may be utilized in the methods andcompositions described below.

As an illustration of one system, adenovirus, a double-stranded DNAvirus, is a well-characterized gene transfer vector for delivery of aheterologous nucleic acid molecule (for a review, see Becker et al.,Meth. Cell Biol. 43:161 (1994); Douglas and Curiel, Science & Medicine4:44 (1997)). The adenovirus system offers several advantages including:(i) the ability to accommodate relatively large DNA inserts, (ii) theability to be grown to high-titer, (iii) the ability to infect a broadrange of mammalian cell types, and (iv) the ability to be used with manydifferent promoters including ubiquitous, tissue specific, andregulatable promoters. In addition, adenoviruses can be administered byintravenous injection, because the viruses are stable in thebloodstream.

Using adenovirus vectors where portions of the adenovirus genome aredeleted, inserts are incorporated into the viral DNA by direct ligationor by homologous recombination with a co-transfected plasmid. In anexemplary system, the essential E1 gene is deleted from the viralvector, and the virus will not replicate unless the E1 gene is providedby the host cell. When intravenously administered to intact animals,adenovirus primarily targets the liver. Although an adenoviral deliverysystem with an E1 gene deletion cannot replicate in the host cells, thehost's tissue will express and process an encoded heterologous protein.Host cells will also secrete the heterologous protein if thecorresponding gene includes a secretory signal sequence. Secretedproteins will enter the circulation from tissue that expresses theheterologous gene (e.g., the highly vascularized liver).

Moreover, adenoviral vectors containing various deletions of viral genescan be used to reduce or eliminate immune responses to the vector. Suchadenoviruses are E1-deleted, and in addition, contain deletions of E2Aor E4 (Lusky et al., J. Virol. 72:2022 (1998); Raper et al., Human GeneTherapy 9:671 (1998)). The deletion of E2b has also been reported toreduce immune responses (Amalfitano et al., J. Virol. 72:926 (1998)). Bydeleting the entire adenovirus genome, very large inserts ofheterologous DNA can be accommodated. Generation of so called “gutless”adenoviruses, where all viral genes are deleted, are particularlyadvantageous for insertion of large inserts of heterologous DNA (for areview, see Yeh. and Perricaudet, FASEB J. 11:615 (1997)).

High titer stocks of recombinant viruses capable of expressing atherapeutic gene can be obtained from infected mammalian cells usingstandard methods. For example, recombinant herpes simplex virus can beprepared in Vero cells, as described by Brandt et al., J. Gen. Virol.72:2043 (1991), Herold et al., J. Gen. Virol. 75:1211 (1994), Visalliand Brandt, Virology 185:419 (1991), Grau et al., Invest. Ophthalmol.Vis. Sci. 30:2474 (1989), Brandt et al., J. Virol. Meth. 36:209 (1992),and by Brown and MacLean (eds.), HSV Virus Protocols (Humana Press1997).

Alternatively, an expression vector comprising a TACI-immunoglobulingene can be introduced into a subject's cells by lipofection in vivousing liposomes. Synthetic cationic lipids can be used to prepareliposomes for in vivo transfection of a gene encoding a marker (Felgneret al., Proc. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey et al., Proc.Nat'l Acad. Sci. USA 85:8027 (1988)). The use of lipofection tointroduce exogenous genes into specific organs in vivo has certainpractical advantages. Liposomes can be used to direct transfection toparticular cell types, which is particularly advantageous in a tissuewith cellular heterogeneity, such as the pancreas, liver, kidney, andbrain. Lipids may be chemically coupled to other molecules for thepurpose of targeting. Targeted peptides (e.g., hormones orneurotransmitters), proteins such as antibodies, or non-peptidemolecules can be coupled to liposomes chemically.

Electroporation is another alternative mode of administration. Forexample, Aihara and Miyazaki, Nature Biotechnology 16:867 (1998), havedemonstrated the use of in vivo electroporation for gene transfer intomuscle.

In general, the dosage of a composition comprising a therapeutic vectorhaving a TACI-immunoglobulin nucleotide acid sequence, such as arecombinant virus, will vary depending upon such factors as thesubject's age, weight, height, sex, general medical condition andprevious medical history. Suitable routes of administration oftherapeutic vectors include intravenous injection, intraarterialinjection, intraperitoneal injection, intramuscular injection,intratumoral injection, and injection into a cavity that contains atumor. As an illustration, Horton et al., Proc. Nat'l Acad. Sci. USA96:1553 (1999), demonstrated that intramuscular injection of plasmid DNAencoding interferon-α produces potent antitumor effects on primary andmetastatic tumors in a murine model.

A composition comprising viral vectors, non-viral vectors, or acombination of viral and non-viral vectors of the present invention canbe formulated according to known methods to prepare pharmaceuticallyuseful compositions, whereby vectors or viruses are combined in amixture with a pharmaceutically acceptable carrier. As noted above, acomposition, such as phosphate-buffered saline is said to be a“pharmaceutically acceptable carrier” if its administration can betolerated by a recipient subject. Other suitable carriers are well-knownto those in the art (see, for example, Remington's PharmaceuticalSciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman's thePharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co.1985)).

For purposes of therapy, a therapeutic gene expression vector, or arecombinant virus comprising such a vector, and a pharmaceuticallyacceptable carrier are administered to a subject in a therapeuticallyeffective amount. A combination of an expression vector (or virus) and apharmaceutically acceptable carrier is said to be administered in a“therapeutically effective amount” if the amount administered isphysiologically significant. An agent is physiologically significant ifits presence results in a detectable change in the physiology of arecipient subject. For example, an agent used to treat inflammation isphysiologically significant if its presence alleviates the inflammatoryresponse. As another example, an agent used to inhibit the growth oftumor cells is physiologically significant if the administration of theagent results in a decrease in the number of tumor cells, decreasedmetastasis, a decrease in the size of a solid tumor, or increasednecrosis of a tumor.

When the subject treated with a therapeutic gene expression vector or arecombinant virus is a human, then the therapy is preferably somaticcell gene therapy. That is, the preferred treatment of a human with atherapeutic gene expression vector or a recombinant virus does notentail introducing into cells a nucleic acid molecule that can form partof a human germ line and be passed onto successive generations (i.e.,human germ line gene therapy).

10. Production of Transgenic Mice

Transgenic mice can be engineered to over-express nucleic acid sequencesencoding TACI-immunoglobulin fusion proteins in all tissues, or underthe control of a tissue-specific or tissue-preferred regulatory element.These over-producers of TACI-immunoglobulin fusion proteins can be usedto characterize the phenotype that results from over-expression, and thetransgenic animals can serve as models for human disease caused byexcess TACI receptor protein. Transgenic mice that over-expressTACI-immunoglobulin fusion proteins also provide model bioreactors forproduction of TACI-immunoglobulin fusion proteins in the milk or bloodof larger animals. Methods for producing transgenic mice are well-knownto those of skill in the art (see, for example, Jacob, “Expression andKnockout of Interferons in Transgenic Mice,” in Overexpression andKnockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111-124(Academic Press, Ltd. 1994), Monastersky and Robl (eds.), Strategies inTransgenic Animal Science (ASM Press 1995), and Abbud and Nilson,“Recombinant Protein Expression in Transgenic Mice,” in Gene ExpressionSystems: Using Nature for the Art of Expression, Fernandez and Hoeffler(eds.), pages 367-397 (Academic Press, Inc. 1999)).

For example, a method for producing a transgenic mouse that expresses anucleic acid sequence that encodes a TACI-immunoglobulin fusion proteincan begin with adult, fertile males (studs) (B6C3f1, 2 to 8 months ofage (Taconic Farms, Germantown, N.Y.)), vasectomized males (duds)(B6D2f1, 2 to 8 months, (Taconic Farms)), prepubescent fertile females(donors) (B6C3f1, 4 to 5 weeks, (Taconic Farms)) and adult fertilefemales (recipients) (B6D2f1, 2 to 4 months, (Taconic Farms)). Thedonors are acclimated for one week and then injected with approximately8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma ChemicalCompany; St. Louis, Mo.) I.P., and 46-47 hours later, 8 IU/mouse ofhuman Chorionic Gonadotropin (hCG (Sigma)) I.P. to inducesuperovulation. Donors are mated with studs subsequent to hormoneinjections. Ovulation generally occurs within 13 hours of hCG injection.Copulation is confirmed by the presence of a vaginal plug the morningfollowing mating.

Fertilized eggs are collected under a surgical scope. The oviducts arecollected and eggs are released into urinanalysis slides containinghyaluronidase (Sigma). Eggs are washed once in hyaluronidase, and twicein Whitten's W640 medium (described, for example, by Menino andO'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote4:129 (1996)) that has been incubated with 5% CO₂, 5% O₂, and 90% N₂ at37° C. The eggs are then stored in a 37° C./5% CO₂ incubator untilmicroinjection.

Ten to twenty micrograms of plasmid DNA containing a TACI-immunoglobulinfusion protein encoding sequence is linearized, gel-purified, andresuspended in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at afinal concentration of 5-10 nanograms per microliter for microinjection.For example, the TACI-immunoglobulin fusion protein encoding sequencescan encode a TACI polypeptide with deletion of amino acid residues 1 to29 and 111 to 154 of SEQ ID NO:2, and an Fc5 immunoglobulin moiety.

Plasmid DNA is microinjected into harvested eggs contained in a drop ofW640 medium overlaid by warm, CO₂-equilibrated mineral oil. The DNA isdrawn into an injection needle (pulled from a 0.75 mm ID, 1 mm ODborosilicate glass capillary), and injected into individual eggs. Eachegg is penetrated with the injection needle, into one or both of thehaploid pronuclei.

Picoliters of DNA are injected into the pronuclei, and the injectionneedle withdrawn without coming into contact with the nucleoli. Theprocedure is repeated until all the eggs are injected. Successfullymicroinjected eggs are transferred into an organ tissue-culture dishwith pre-gassed W640 medium for storage overnight in a 37° C./5% CO₂incubator.

The following day, two-cell embryos are transferred into pseudopregnantrecipients. The recipients are identified by the presence of copulationplugs, after copulating with vasectomized duds. Recipients areanesthetized and shaved on the dorsal left side and transferred to asurgical microscope. A small incision is made in the skin and throughthe muscle wall in the middle of the abdominal area outlined by theribcage, the saddle, and the hind leg, midway between knee and spleen.The reproductive organs are exteriorized onto a small surgical drape.The fat pad is stretched out over the surgical drape, and a babyserrefine (Roboz, Rockville, Md.) is attached to the fat pad and lefthanging over the back of the mouse, preventing the organs from slidingback in.

With a fine transfer pipette containing mineral oil followed byalternating W640 and air bubbles, 12-17 healthy two-cell embryos fromthe previous day's injection are transferred into the recipient. Theswollen ampulla is located and holding the oviduct between the ampullaand the bursa, a nick in the oviduct is made with a 28 g needle close tothe bursa, making sure not to tear the ampulla or the bursa.

The pipette is transferred into the nick in the oviduct, and the embryosare blown in, allowing the first air bubble to escape the pipette. Thefat pad is gently pushed into the peritoneum, and the reproductiveorgans allowed to slide in. The peritoneal wall is closed with onesuture and the skin closed with a wound clip. The mice recuperate on a37° C. slide warmer for a minimum of four hours.

The recipients are returned to cages in pairs, and allowed 19-21 daysgestation. After birth, 19-21 days postpartum is allowed before weaning.The weanlings are sexed and placed into separate sex cages, and a 0.5 cmbiopsy (used for genotyping) is snipped off the tail with cleanscissors.

Genomic DNA is prepared from the tail snips using, for example, a QIAGENDNEASY kit following the manufacturer's instructions. Genomic DNA isanalyzed by PCR using primers designed to amplify a nucleic acidsequence encoding a TACI-immunoglobulin fusion protein or a selectablemarker gene that was introduced in the same plasmid. After animals areconfirmed to be transgenic, they are back-crossed into an inbred strainby placing a transgenic female with a wild-type male, or a transgenicmale with one or two wild-type female(s). As pups are born and weaned,the sexes are separated, and their tails snipped for genotyping.

To check for expression of a transgene in a live animal, a partialhepatectomy is performed. A surgical prep is made of the upper abdomendirectly below the zyphoid process. Using sterile technique, a small1.5-2 cm incision is made below the sternum and the left lateral lobe ofthe liver exteriorized. Using 4-0 silk, a tie is made around the lowerlobe securing it outside the body cavity. An atraumatic clamp is used tohold the tie while a second loop of absorbable Dexon (American Cyanamid;Wayne, N.J.) is placed proximal to the first tie. A distal cut is madefrom the Dexon tie and approximately 100 mg of the excised liver tissueis placed in a sterile petri dish. The excised liver section istransferred to a 14 ml polypropylene round bottom tube and snap frozenin liquid nitrogen and then stored on dry ice. The surgical site isclosed with suture and wound clips, and the animal's cage placed on a37° C. heating pad for 24 hours post operatively. The animal is checkeddaily post operatively and the wound clips removed 7-10 days aftersurgery. The expression level of TACI-immunoglobulin fusion protein mRNAis examined for each transgenic mouse using an RNA solutionhybridization assay or polymerase chain reaction.

Using the general approach described above, transgenic mice have beenproduced that express significant levels of TACI-immunoglobulin fusionprotein in milk. In this particular case, the TACI-immunoglobulin fusionprotein encoding sequence encoded a TACI polypeptide with deletion ofamino acid residues 1 to 29 and 111 to 154 of SEQ ID NO:2, and an Fc5immunoglobulin moiety.

The present invention, thus generally described, will be understood morereadily by reference to the following examples, which are provided byway of illustration and is not intended to be limiting of the presentinvention.

Experimental Example 1

Construction of Nucleic Acid Molecules that Encode TACI-Fc Proteins

Nucleic acid molecules encoding human TACI were obtained during theexpression cloning of the receptors for ZTNF4 as described by Gross etal., Nature 404:995 (2000). The coding sequences contained in theTACI-Fc expression constructs were generated by overlap PCR, usingstandard techniques (see, for example, Horton et al., Gene 77:61(1989)). Human TACI cDNA and Fc cDNA were used as starting templates forthe PCR amplifications. PCR primers were designed to yield the desiredcoding sequence 5′ and 3′ ends and to introduce restriction enzymerecognition sites to facilitate insertion of these coding sequences intothe expression vectors. The TACI-Fc coding sequences were inserted intoexpression vectors that included a functional murine dihydrofolatereductase gene. One expression vector also contained a cytomegaloviruspromoter to direct the expression of the recombinant protein transgene,an immunoglobulin intron, a tissue plasminogen activator signalsequence, an internal ribosome entry sequence, a deleted CD8 cistron forsurface selection of transfected cells, and yeast expression elementsfor growth of the plasmid in yeast cells.

One approach that was used to produce TACI-Fc fusion proteins isillustrated by the method used to construct TACI-Fc4. Other TACI-Fcfusion proteins were produced by inserting nucleotide sequences thatencode a TACI-Fc fusion protein into a mammalian expression vector, andintroducing that expression vector into mammalian cells.

A. Ig γ1 Fc4 Fragment Construction

To prepare the TACI-Fc4 fusion protein, the Fc region of human IgG1 (thehinge region and the CH₂ and CH₃ domains) was modified to remove Fcγ1receptor (FcγRI) and complement (C1q) binding functions. This modifiedversion of human IgG1 Fc was designated “Fc4.”

The Fc region was isolated from a human fetal liver library (Clontech)PCR using oligo primers 5′ ATCAGCGGAA TTCAGATCTT CAGACAAAAC TCACACATGCCCAC 3′ (SEQ ID NO:7) and 5′ GGCAGTCTCT AGATCATTTA CCCGGAGACA GGGAG 3′(SEQ ID NO:8). Mutations within the Fc region were introduced by PCR toreduce FcγRI binding. The FcγRI binding site (Leu-Leu-Gly-Gly; aminoacid residues 38 to 41 of SEQ ID NO:6, which correspond to EU indexpositions 234 to 237) was mutated to Ala-Glu-Gly-Ala to reduce FcγR1binding (see, for example, Duncan et al., Nature 332:563 (1988); Baum etal., EMBO J. 13:3992 (1994)). Oligonucleotide primers 5′ CCGTGCCCAGCACCTGAAGC CGAGGGGGCA CCGTCAGTCT TCCTCTTCCC C 3′ (SEQ ID NO:9) and 5′GGATTCTAGA TTATTTACCC GGAGACAGGG A 3′ (SEQ ID NO:10) were used tointroduce the mutation. To a 50 μl final volume was added 570 ng of IgFctemplate, 5 μl of 10× Pfu reaction Buffer (Stratagene), 8 μl of 1.25 mMdNTPs, 31 μl of distilled water, 2 μl of 20 mM oligonucleotide primers.An equal volume of mineral oil was added and the reaction was heated to94° C. for 1 minute. Pfu polymerase (2.5 units, Stratagene) was addedfollowed by 25 cycles at 94° C. for 30 seconds, 55° C. for 30 seconds,72° C. for 1 minute followed by a 7 minute extension at 72° C. Thereaction products were fractioned by electrophoresis, and the bandcorresponding to the predicted size of about 676 base pairs wasdetected. This band was excised from the gel and recovered using aQIAGEN QIAquick™ Gel Extraction Kit (Qiagen) according to themanufacturer's instructions.

PCR was also used to introduce a mutation of Ala to Ser (amino acidresidue 134 of SEQ ID NO:6, which corresponds to EU index position 330)and Pro to Ser (amino acid residue 135 of SEQ ID NO:6, which correspondsto EU index position 331) to reduce complement C1q binding or complementfixation (Duncan and Winter, Nature 332:788 (1988)). Two first roundreactions were performed using the FcγRI binding side-mutated IgFcsequence as a template. To a 50 μl final volume was added 1 μl of FcγRIbinding site mutated IgFc template, 5 μl of 10× Pfu Reaction Buffer(Stratagene), 8 μl of 1.25 mM dNTPs, 31 of μl distilled water, 2 μl of20 mM 5′ GGTGGCGGCT CCCAGATGGG TCCTGTCCGA GCCCAGATCT TCAGACAAAA CTCAC 3′(SEQ ID NO:11), a 5′ primer beginning at nucleotide 36 of SEQ ID NO:5,and 2 μl of 20 mM 5′ TGGGAGGGCT TTGTTGGA 3′ (SEQ ID NO:12), a 3′ primerbeginning at the complement of nucleotide 405 of SEQ ID NO:5. The secondreaction contained 2 μl each of 20 mM stocks of oligonucleotide primers5′ TCCAACAAAG CCCTCCCATC CTCCATCGAG AAAACCATCT CC 3′ (SEQ ID NO:13), a5′ primer beginning at nucleotide 388 of SEQ ID NO:5 and 5′ GGATGGATCCATGAAGCACC TGTGGTTCTT CCTCCTGCTG GTGGCGGCTC CCAGATG 3′ (SEQ ID NO:14), a3′ primer, to introduce the Ala to Ser mutation, XbaI restriction siteand stop codon. An equal volume of mineral oil was added and thereactions were heated to 94° C. for 1 minute. Pfu polymerase (2.5 units,Stratagene) was added followed by 25 cycles at 94° C. for 30 seconds,55° C. for 30 seconds, 72° C. for 2 minutes followed by a 7 minuteextension at 72° C. The reaction products were fractionated byelectrophoresis, and bands corresponding to the predicted sizes, about370 and about 395 base pairs respectively, were detected. The bands wereexcised from the gel and extracted using a QIAGEN QIAquick™ GelExtraction Kit (Qiagen) according to the manufacturer's instructions.

A second round reaction was performed to join the above fragments andadd the 5′ BamHI restriction site and a signal sequence from the humanimmunoglobulin JBL 2′C_(L) heavy chain variable region (Cogne et al.,Eur. J. Immunol. 18:1485 (1988)). To a 50 μl final volume was added 30μl of distilled water, 8 μl of 1.25 mM dNTPs, 5 μl of 10× Pfu polymerasereaction buffer (Stratagene) and 1 μl each of the two first two PCRproducts. An equal volume of mineral oil was added and the reaction washeated to 94° C. for 1 minute. Pfu polymerase (2.5 units, Stratagene)was added followed by 5 cycles at 94° C. for 30 seconds, 55° C. for 30seconds, and 72° C. for 2 minutes. The temperature was again brought to94° C. and 2 μl each of 20 mM stocks of 5′ GGATGGATCC ATGAAGCACCTGTGGTTCTT CCTCCTGCTG GTGGCGGCTC CCAGATG 3′ (SEQ ID NO:14), a 5′ primerbeginning at nucleotide 1 of SEQ ID NO:5, and 5′ GGATTCTAGA TTATTTACCCGGAGACAGGG A 3′ (SEQ ID NO:10) were added followed by 25 cycles at 94°C. for 30 seconds, 55° C. for 30 seconds and 72° C. for 2 minutes, and afinal 7 minute extension at 72° C. A portion of the reaction wasvisualized using gel electrophoresis. A 789 base pair band correspondingthe predicted size was detected.

B. TACI-Fc4 Expression Vector Construction

Expression plasmids comprising a coding region for TACI-Fc4 fusionprotein were constructed via homologous recombination in yeast. Afragment of TACI cDNA was isolated using PCR that included thepolynucleotide sequence from nucleotide 14 to nucleotide 475 of SEQ IDNO:1. The two primers used in the production of the TACI fragment were:(1) a primer containing 40 base pairs of the 5′ vector flanking sequenceand 17 base pairs corresponding to the amino terminus of the TACIfragment (5′ CTCAGCCAGG AAATCCATGC CGAGTTGAGA CGCTTCCGTA GAATGAGTGGCCTGGGCCG 3; SEQ ID NO:15); (2) 40 base pairs of the 3′ endcorresponding to the flanking Fc4 sequence and 17 base pairscorresponding to the carboxyl terminus of the TACI fragment (5′GCATGTGTGA GTTTTGTCTG AAGATCTGGG CTCCTTCAGC CCCGGGAG 3; SEQ ID NO:16).To a 100 μl final volume was added 10 ng of TACI template, 10 μl of 10×Taq polymerase Reaction Buffer (Perkin Elmer), 8 μl of 2.5 nM dNTPs, 78μl of distilled water, 2 μl each of 20 mM stocks of the oligonucleotideprimers, and Taq polymerase (2.5 units, Life Technology). An equalvolume of mineral oil was added and the reaction was heated to 94° C.for 2 minutes, followed by 25 cycles at 94° C. for 30 seconds, 65° C.for 30 seconds, 65° C. for 30 seconds, 72° C. for 1 minute followed by a5 minute extension at 72° C.

The fragment containing the cDNA encoding the Fc4 fragment wasconstructed in a similar manner. The two primers used in the productionof the Fc4 fragment were (upstream and downstream), an oligonucleotideprimer containing 40 base pairs of the 5′ TACI flanking sequence and 17base pairs corresponding to the amino terminus of the Fc4 fragment (5′GCACAGAGGC TCAGAAGCAA GTCCAGCTCT CCCGGGGCTG AAGGAGCCCA GATCTTCAGA 3; SEQID NO:17); and an oligonucleotide primer containing 40 base pairs of the3′ end corresponding to the flanking vector sequence and 17 base pairscorresponding to the carboxyl terminus of the Fc4 fragment (5′GGGGTGGGTA CAACCCCAGA GCTGTTTTAA TCTAGATTAT TTACCCGGAG ACAGGG 3; SEQ IDNO:18). To a 100 μl final volume was added 10 ng of Fc4 templatedescribed above, 10 μl 10× Taq polymerase Reaction Buffer (PerkinElmer), 8 μl of 2.5 nM dNTPs, 78 μl of distilled water, 2 μl each of 20mM stocks of the oligonucleotides, and Taq polymerase (2.5 units, LifeTechnology). An equal volume of mineral oil was added and the reactionwas heated to 94° C. for 2 minutes, then 25 cycles at 94° C. for 30seconds, 65° C. for30 seconds, 72° C. for 1 minute followed by a 5minute extension at 72° C.

Ten microliters of each of the 100 μl PCR reactions described above wererun on a 0.8% LMP agarose gel (Seaplaque GTG) with 1×TBE buffer foranalysis. The remaining 90 μl of each PCR reaction was precipitated withthe addition of 5 μl of 1 M sodium chloride and 250 μl of absoluteethanol. The plasmid pZMP6 was cleaved with SmaI to linearize it at thepolylinker. Plasmid pZMP6 was derived from the plasmid pCZR199 (AmericanType Culture Collection, Manassas, Va., ATCC#98668) and is a mammalianexpression vector containing an expression cassette having thecytomegalovirus immediate early promoter, a consensus intron from thevariable region of mouse immunoglobulin heavy chain locus, multiplerestriction sites for insertion of coding sequences, a stop codon and ahuman growth hormone terminator. The plasmid also has an E. coli originof replication, a mammalian selectable marker expression unit having anSV40 promoter, enhancer and origin of replication, a dihydrofolatereductase gene and the SV40 terminator. The vector pZMP6 was constructedfrom pCZR199 by replacement of the metallothionein promoter with thecytomegalovirus immediate early promoter, and the Kozac sequences at the5′ end of the open reading frame.

One hundred microliters of competent yeast cells (S. cerevisiae) werecombined with 10 μl containing approximately 1 μg of the TACIextracellular domain and the Fc4 PCR fragments, and 100 ng of SmaIdigested pZMP6 vector and transferred to a 0.2 cm electroporationcuvette. The yeast/DNA mixtures were electropulsed at 0.75 kV (5 kV/cm),∞ ohms, 25 μF. To each cuvette was added 600 μl of 1.2 M sorbitol andthe yeast were plated in two 300 μl aliquots onto to URA-D plates andincubated at 30° C.

After about 48 hours, the Ura+yeast transformants from a single platewere resuspended in 1 ml of water and spun briefly to pellet the yeastcells. The cell pellet was resuspended in 1 ml of lysis buffer (2%Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris, pH 8.0, 1 mM EDTA). Fivehundred microliters of the lysis mixture was added to an Eppendorf tubecontaining 300 μl acid washed glass beads and 200 μl phenol-chloroform,vortexed for 1 minute intervals two or three times, followed by a 5minute spin in a Eppendorf centrifuge at maximum speed. Three hundredmicroliters of the aqueous phase was transferred to a fresh tube, andthe DNA precipitated with 600 μl of ethanol, followed by centrifugationfor 10 minutes at 4° C. The DNA pellet was resuspended in 100 μl ofwater.

Transformation of electrocompetent E. coli cells (DH10B, GibcoBRL) wasperformed with 0.5-2 ml yeast DNA prep and 40 μl of DH10B cells. Thecells were electropulsed at 2.0 kV, 25 mF and 400 ohms Followingelectroporation, 1 ml of SOC (2% Bacto-Tryptone (Difco, Detroit, Mich.),0.5% yeast extract (Difco), 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, 10 mMMgSO₄, 20 mM glucose) were plated in 250 μl aliquots on four LB AMPplates (LB broth (Lennox), 1.8% Bacto-Agar (Difco), 100 mg/LAmpicillin).

Individual clones harboring the correct expression construct forTACI-Fc4 were identified by restriction digest to verify the presence ofthe insert and to confirm that the various DNA sequences have beenjoined correctly to one another. The insert of positive clones weresubjected to sequence analysis. Larger scale plasmid DNA is isolatedusing the Qiagen Maxi kit (Qiagen) according to manufacturer'sinstructions.

C. Construction of Fc5, Fc6, and Fc7

In Fc5, the Arg residue at EU index position 218 was changed back to aLys residue. Wild-type human Ig γ1 contains a lysine at this position.Briefly, nucleic acid molecules encoding Fc5 were produced usingoligonucleotide primers 5′ GAGCCCAAATCTTCAGACAAAACTCACACATGCCCA 3′ (SEQID NO:19) and 5′ TAATTGGCGCGCCTCTAGATTATTTACCCGGAGACA 3′ (SEQ ID NO:20).The conditions of the PCR amplification were as follows. To a 50 μlfinal volume was added 236 ng of Fc4 template, 5 μl of 10 Pfu reactionBuffer (Stratagene), 4 μl of 2.5 mM dNTPs, 1 μl of 20 μM of each of theoligonucleotides, and 1 μl of Pfu polymerase (2.5 units, Stratagene).The amplification thermal profile consisted of 94° C. for 2 minutes, 5cycles at 94° C. for 15 seconds, 42° C. for 20 seconds, 72° C. for 45seconds, 20 cycles at 94° C. for 15 seconds, 72° C. for 1 minute 20seconds, followed by a 7 minute extension at 72° C. The reaction productwas fractionated by agarose gel electrophoresis, and the bandcorresponding to the predicted size of about 718 base pairs wasdetected. The band was excised from the gel and recovered using a QIAGENQIAquick Gel Extraction Kit (Qiagen) according to the manufacturer'sinstructions.

Fc6 is identical to Fc5 except that the carboxyl terminal lysine codonhas been eliminated. As in Fc4 and Fc5 above, the stop codon in the Fc6sequence was changed to TAA. Fc6 was generated from template DNA thatencoded Fc5 using oligonucleotide primers 5′ GAGCCCAAAT CTTCAGACAAAACTCACACA TGCCCA 3′ (SEQ ID NO:19) and 5′ GGCGCGCCTC TAGATTAACCCGGAGACAGG GAGAGGC 3′ (SEQ ID NO:21).

Fc7 is identical to the wild-type γ1 Fc except for an amino acidsubstitution at EU index position Asn 297 located in the C_(H)2 domain.Asn 297 was mutated to a Gln residue to prevent the attachment ofN-linked carbohydrate at that residue position. As above, the stop codonin the Fc7 sequence was changed to TAA. Fc7 was generated by overlap PCRusing a wild-type human IgGγ1 Fc cDNA as the template andoligonucleotide primers 5′ GAGCCCAAATCTTGCGACAAAACTCACA 3′ (SEQ IDNO:22) and 5′ GTACGTGCTTTGGTACIGCTCCTCCCGCGGCTT 3′ (SEQ ID NO:23) togenerate the 5′ half of Fc7, and oligonucleotide primers 5′CAGTACCAAAGCACGTACCGTGTGGTCA 3′ (SEQ ID NO:24) and 5′TAATTGGCGCGCCTCTAGATTATTTACCCGGAGACA 3′ (SEQ ID NO:20) to generate the3′ half of Fc7. The two PCR products were combined and amplified usingoligonucleotide primers 5′ GAGCCCAAATCTTGCGACAAAACTCACA 3′ (SEQ IDNO:22) and 5′ TAATTGGCGCGCCTCTAGATTATTTACCCGGAGACA 3′ (SEQ ID NO:20).

All the resultant PCR products were gel purified, cloned, and verifiedby DNA sequence analysis.

D. Construction of Amino-Truncated TACI-Fc Fusion Proteins

Four amino terminal truncated versions of TACI-Fc were generated. Allfour had a modified human tissue plasminogen activator signal sequence(SEQ ID NO:25) fused to amino acid residue number 30 of SEQ ID NO:2.However, the four proteins differed in the location of point in whichthe Fc5 was fused to the TACI amino acid sequence of SEQ ID NO:2. Table3 outlines the structures of the four fusion proteins.

TABLE 3 TACI Fusion Proteins Designation of TACI-Fc TACI amino acidresidues TACI(d1-29)-Fc5 30 to 154 of SEQ ID NO: 2 TACI(d1-29,d107-154)-Fc5 30 to 106 of SEQ ID NO: 2 TACI(d1-29, d111-154)-Fc5 30 to110 of SEQ ID NO: 2 TACI(d1-29, d120-154)-Fc5 30 to 119 of SEQ ID NO: 2

Protein encoding expression cassettes were generated by overlap PCRusing standard techniques (see, for example, Horton et al., Gene 77:61(1989)). A nucleic acid molecule encoding TACI and a nucleic acidmolecule encoding Fc5 were used as PCR templates. Oligonucleotideprimers are identified in Tables 4 and 5.

TABLE 4 Oligonucleotide Primers Used to Produce TACI Fusion ProteinsOligonucleotide Designations Designation of TACI-Fc 5′ TACI 3′ TACI 5′Fc5 3′ Fc5 TACI(d1-29)-Fc5 ZC24,903 ZC24,955 ZC24,952 ZC24,946TACI(d1-29, d107-154)- ZC24,903 ZC24,951 ZC24,949 ZC24,946 Fc5TACI(d1-29, d111-154)- ZC24,903 ZC28,978 ZC28,979 ZC24,946 Fc5TACI(d1-29, d120-154)- ZC24,903 ZC28,981 ZC28,980 ZC24,946 Fc5

TABLE 5 Oligonucleotide Sequences Primer Nucleotide Sequence SEQ ID NO.ZC24,903 5′ TATTAGGCCGGCCACCATGGATGCAATGA 3′ 40 ZC24,9555′ TGAAGATTTGGGCTCCTTGAGACCTGGGA 3′ 41 ZC24,9525′ TCCCAGGTCTCAAGGAGCCCAAATCTTCA 3′ 42 ZC24,9465′ TAATTGGCGCGCCTCTAGATTATTTACCCGGAGACA 3′ 20 ZC24,9515′ TGAAGATTTGGGCTCGTTCTCACAGAAGTA 3′ 43 ZC24,9495′ ATACTTCTGTGAGAACGAGCCCAAATCTTCA 3′ 44 ZC28,9785′ TTTGGGCTCGCTCCTGAGCTTGTTCTCACA 3′ 45 ZC28,9795′ CTCAGGAGCGAGCCCAAATCTTCAGACA 3′ 46 ZC28,9815′ TTTGGGCTCCCTGAGCTCTGGTGGAA 3′ 47 ZC28,9805′ GAGCTCAGGGAGCCCAAATCTTCAGACA 3′ 48

The first round of PCR amplifications consisted of two reactions foreach of the four amino terminal truncated versions. The two reactionswere performed separately using the 5′and 3′ TACI oligonucleotides inone reaction, and the 5′ and 3′ Fc5 oligonucleotides in another reactionfor each version. The conditions of the first round PCR amplificationwere as follows. To a 25 μl final volume was added approximately 200 ngtemplate DNA, 2.5 μl 10× Pfu reaction Buffer (Stratagene), 2 μl of 2.5mM dNTPs, 0.5 μl of 20 μM each 5′ oligonucleotide and 3′oligonucleotide, and 0.5 μl Pfu polymerase (2.5 units, Stratagene). Theamplification thermal profile consisted of 94° C. for 3 minutes, 35cycles at 94° C. for 15 seconds, 50° C. for 15 seconds, 72° C. for 2minutes, followed by a 2 minute extension at 72° C. The reactionproducts were fractionated by agarose gel electrophoresis, and the bandscorresponding to the predicted sizes were excised from the gel andrecovered using a QIAGEN QIAQUICK Gel Extraction Kit (Qiagen), accordingto the manufacturer's instructions.

The second round of PCR amplification, or overlap PCR amplificationreaction, was performed using the gel purified fragments from the firstround PCR as DNA template. The conditions of the second round PCRamplification were as follows. To a 25 μl final volume was addedapproximately 10 ng template DNA each of the TACI fragment and the Fc5fragment, 2.5 μl 10× Pfu reaction Buffer (Stratagene), 2 μl of 2.5 mMdNTPs, 0.5 μl of 20 μM each ZC24,903 (SEQ ID NO:40) and ZC24,946 (SEQ IDNO:20) and 0.5 μl Pfu polymerase (2.5 units, Stratagene). Theamplification thermal profile consisted of 94° C. for 1 minute, 35cycles at 94° C. for 15 seconds, 55° C. for 15 seconds, 72° C. for 2minutes, followed by a 2 minute extension at 72° C. The reactionproducts were fractionated by agarose gel electrophoresis, and the bandscorresponding to the predicted sizes were excised from the gel andrecovered using a QIAGEN QIAQUICK Gel Extraction Kit (Qiagen), accordingto the manufacturer's instructions.

Each of the four versions of the amino terminal truncated TACI-Fc PCRproducts were separately cloned using Invitrogen's ZEROBLUNT TOPO PCRCloning Kit following the manufacturer's recommended protocol. Table 6identifies the nucleotide and amino acid sequences of these TACI-Fcconstructs.

TABLE 6 Sequences of TACI-Fc Variants SEQ ID Nos. Designation of TACI-FcNucleotide Amino Acid TACI(d1-29)-Fc5 49 50 TACI(d1-29, d107-154)-Fc5 5152 TACI(d1-29, d111-154)-Fc5 53 54 TACI(d1-29, d120-154)-Fc5 55 56

After the nucleotide sequences were verified, plasmids comprising eachof the four versions of the amino terminal truncated TACI-Fc fusionswere digested with FseI and AscI to release the amino acid encodingsegments. The FseI-AscI fragments were ligated into a mammalianexpression vector containing a CMV promoter and an SV40 poly A segment.Expression vectors were introduced into Chinese hamster ovary cells asdescribed below.

Example 2 Production of TACI-Fc Proteins by Chinese Hamster Ovary Cells

The TACI-Fc expression constructs were used to transfect, viaelectroporation, suspension-adapted Chinese hamster ovary (CHO) DG44cells grown in animal protein-free medium (Urlaub et al., Som. Cell.Molec. Genet. 12:555 (1986)). CHO DG44 cells lack a functionaldihydrofolate reductase gene due to deletions at both dihydrofolatereductase chromosomal locations. Growth of the cells in the presence ofincreased concentrations of methotrexate results in the amplification ofthe dihydrofolate reductase gene, and the linked recombinantprotein-encoded gene on the expression construct.

CHO DG44 cells were passaged in PFCHO media (JRH Biosciences, Lenexa,Kans.), 4 mM L-Glutamine (JRH Biosciences), and 1×hypothanxine-thymidine supplement (Life Technologies), and the cellswere incubated at 37° C. and 5% CO₂ in Corning shake flasks at 120 RPMon a rotating shaker platform. The cells were transfected separatelywith linearized expression plasmids. To ensure sterility, a singleethanol precipitation step was performed on ice for 25 minutes bycombining 200 μg of plasmid DNA in an Eppendorf tube with 20 μl ofsheared salmon sperm carrier DNA (5′→3′ Inc. Boulder, Colo., 10 mg/ml),22 μl of 3M NaOAc (pH 5.2), and 484 μl of 100% ethanol (Gold ShieldChemical Co., Hayward, Calif.). After incubation, the tube wascentrifuged at 14,000 RPM in a microfuge placed in a 4° C. cold room,the supernatant removed and the pellet washed twice with 0.5 ml of 70%ethanol and allowed to air dry.

The CHO DG44 cells were prepared while the DNA pellet was drying bycentrifuging 10⁶ total cells (16.5 ml) in a 25 ml conical centrifugetube at 900 RPM for 5 minutes. The CHO DG44 cells were resuspended intoa total volume of 300 μl of PFCHO growth media, and placed in aGene-Pulser Cuvette with a 0.4 cm electrode gap (Bio-Rad). The DNA,after approximately 50 minutes of drying time, was resuspended into 500μl of PFCHO growth media and added to the cells in the cuvette so thatthe total volume did not exceed 800 μl and was allowed to sit at roomtemperature for 5 minutes to decrease bubble formation. The cuvette wasplaced in a BioRad Gene Pulser II unit set at 0.296 kV (kilovolts) and0.950 HC (high capacitance) and electroporated immediately.

The cells were incubated 5 minutes at room temperature before placementin 20 ml total volume of PFCHO media in a CoStar T-75 flask. The flaskwas placed at 37° C. and 5% CO₂ for 48 hours when the cells were thencounted by hemocytometer utilizing trypan blue exclusion and put intoPFCHO selection media without hypothanxine-thymidine supplement andcontaining 200 mM methotrexate (Cal Biochem).

Upon recovery of the methotrexate selection process, the conditionedmedia containing the secreted TACI-Fc proteins were examined by WesternBlot analysis.

Example 3 Structural Analysis of TACI-Fc Proteins

In certain cases, TACI-Fc fusion proteins were partially purified beforeanalysis. Conditioned medium from Chinese hamster ovary cultures wassterile-filtered through a 0.22 μm filter and the TACI-Fc protein wascaptured on a protein A column. The protein A-bound material was elutedand passed over an S-200 size exclusion column for final purification.

Western blot analysis was performed on both conditioned cell medium andpurified protein to assess the structural stability of the TACI-Fcproteins. Briefly, protein or supernatant samples were transferred tonitrocellulose membranes and the TACI-Fc proteins were detected usingperoxidase conjugated goat anti-mouse IgG2a (Boehringer Mannheim), orperoxidase conjugated goat anti-human IgG Fc specific antisera (Pierce).

Amino terminal amino acid sequence analyses were performed on Models476A and 494 Protein Sequencer Systems from Perkin Elmer AppliedBiosystems Division (Foster City, Calif.). Data analysis was performedwith Applied Biosystems Model 610A Data Analysis System for ProteinSequencing, version 2.1a (Applied Biosystems, Inc.). Most supplies andreagents used were from Applied Biosystems, Inc.

Example 4 Functional Analysis of TACI-Fc Proteins

Two approaches were used to examine the binding characteristics of fourTACI-Fc proteins with ZTNF4. One approach measured the ability of theTACI-Fc constructs to compete with TACI-coated plates for binding of¹²⁵I-labeled ZTNF4. In the second approach, increasing concentrations of¹²⁵I labeled ZTNF4 were incubated with each of the TACI-Fc constructs,and the radioactivity associated with precipitated ZTNF4-TACI-Fccomplexes was determined. The TACI-Fc fusion proteins had TACI moietiesthat lacked the first 29 amino acid residues of the amino acid sequenceof SEQ ID NO:2. One of the fusion proteins had a TACI moiety with anintact stalk region (TACI (d1-29)-Fc5), whereas three of the TACI-Fcfusion proteins had TACI moieties with various deletions in the stalkregion (TACI (d1-29, d107-154)-Fc5; TACI (d1-29, d111-154)-Fc5; TACI(d1-29, d120-154)-Fc5).

A. Competitive Binding Assay

ZTNF4 was radiodinated with Iodobeads (Pierce), following standardmethods. Briefly, 50 μg of the ZTNF4 was iodinated with 4 mCi of ¹²⁵Iusing a single Iodobead. The reaction was quenched with a 0.25% solutionof bovine serum albumin, and the free ¹²⁵I was removed by gel filtrationusing a PD-10 column (Pierce). The specific radioactivity of ¹²⁵I-ZTNF4preparations was determined by trichloroacetic acid precipitation beforeand after the desalting step.

An N-terminal fragment of the TACI receptor, designated as “TACI-N,” wasadded to 96-well plates (100 μl at 0.1 μg/ml), and incubated overnightat 4° C. The plates were washed, blocked with Superblock (Pierce), andwashed again. The TACI-Fc constructs, at various concentrations rangingfrom 0 to 11.5 ng/ml, were mixed with a fixed concentration of¹²⁵I-ZTNF4 (20 ng/ml), and incubated for 2 hours at 37° C. on the platecoated with TACI-N. Controls contained either TACI-N in solution, orlacked TACI-Fc. After incubation, the plates were washed and counted.Each determination was performed in triplicate.

The results showed that all TACI-Fc constructs inhibited ¹²⁵I-ZTNF4binding completely at concentrations of about 100 ng/ml or greater. TheTACI-Fc proteins, TACI (d1-29)-Fc5, TACI (d1-29, d111-154)-Fc5, and TACI(d1-29, d120-154)-Fc5, were more effective inhibitors than the TACI-Fcconstruct, TACI (d1-29, d107-154)-Fc5. An Fc fragment alone did notinhibit binding. IC₅₀ values were calculated for each construct in threeexperiments. The average values for the constructs were: TACI(d1-29)-Fc5: 2.07 nM; TACI (d1-29, d107-154)-Fc5: 4.95 nM; TACI (d1-29,d111-154)-Fc5: 2.31 nM; and TACI (d1-29, d120-154)-Fc5: 2.21 nM.

B. Solution Binding Assay

At a concentration of 0.05 nM, each TACI-Fc construct was incubated with0.4 pM to 1.5 nM ¹²⁵I-ZTNF4 for 30 minutes at room temperature in atotal volume of 0.25 ml/tube. A Pansorbin (Staph A) suspension was addedto each tube, and after 15 minutes, the samples were centrifuged, washedtwice, and the pellets counted. Nonspecific binding was determined bythe addition of 130 nM unlabeled ZTNF4 to the ¹²⁵I-ZTNF4/TACI-Fc mix.Specific binding was calculated by subtracting the cpm bound in thepresence of unlabeled ZTNF4 from the total cpm bound at eachconcentration of ¹²⁵I-ZTNF4. Each determination was performed intriplicate. Binding constants were calculated using GraphPad Prismsoftware (MacIntosh v. 3.0).

FIG. 4 illustrates the specific binding of ¹²⁵I-ZTNF4 to the variousTACI-Fc constructs. Binding appeared to approach saturation with eachconstruct, and was significantly higher for constructs TACI (d1-29)-Fc5,TACI (d1-29, d111-154)-Fc5, TACI (d1-29, d120-154)-Fc5, as compared withthe binding of TACI (d1-29, d107-154)-Fc5. Bmax and Kd values werecalculated, and the results are summarized in Table 7.

TABLE 7 Binding of ¹²⁵I-ZTNF4 to TACI-Fc Constructs TACI-Fc Construct Kd(nM) Bmax (nM) TACI (d1-29)-Fc5 0.134 0.023 TACI (d1-29, d107-154)-Fc50.121 0.010 TACI (d1-29, d111-154)-Fc5 0.115 0.018 TACI (d1-29,d120-154)-Fc5 0.092 0.021

Example 5 Measurement of Circulating ZTNF4

Levels of ZTNF4 in individuals with a disease condition (such as SLE,rheumatoid arthritis for example) relative to normal individuals weredetermined using an electrochemiluminescence assay. A standard curveprepared from soluble, human ZTNF4 at 10 ng/ml, 1 ng/ml, 0.1 ng/ml, 0.01ng/ml and 0 ng/ml was prepared in ORIGIN buffer (Igen, Gaithersburg,Md.). Serum samples were diluted in ORIGIN buffer. The standards andsamples were incubated at room temperature for two hours withbiotinylated rabbit anti-human ZTNF4-NF BV antibody diluted to 1 μg/mlin Origin Assay Buffer (IGEN) and ruthenylated rabbit anti-humanZTNF4-NF BV polyclonal antibody diluted to 1 μg/ml in Origin AssayBuffer (IGEN). Following the incubation the samples were vortexed and0.4 mg/ml streptavidin Dynabeads (Dynal, Oslo, Norway) were added toeach of the standards and samples at 50 μl/tube and incubated for 30minutes at room temperature. Samples were then vortexed and samples wereread on an Origin Analyzer (Igen) according to manufacturer'sinstructions. The Origin assay is based on electrochemiluminescence andproduces a readout in ECL. In one study, an elevated level of ZTNF4 wasdetected in the serum samples from both NZBWF1/J, and MRL/Mpj-Fas^(lpr)mice, which have progressed to advanced stages of glomerulonephritis andautoimmune disease.

The ORIGIN ASSAY was also used to measure levels of ZTNF4 in the bloodof SLE patients, relative to circulating levels in normal individuals. Astandard curve prepared from soluble, human ZTNF4 at 10 ng/ml, 1 ng/ml,0.1 ng/ml, 0.01 ng/ml and 0 ng/ml was prepared in ORIGIN buffer (Igen).All patient samples were run in triplicate with a 25 μl final volume.The standards and samples were incubated at room temperature for twohours with a capture antibody, biotinylated rabbit anti-human ZTNF4-NFBV polyclonal antibody, diluted to 1 μg/ml in Origin Assay Buffer (IGEN)and a detection antibody, ruthenylated rabbit anti-human ZTNF4-NF BVpolyclonal antibody, diluted to 1 μg/ml in Origin Assay Buffer (IGEN).Following the incubation the samples were vortexed, and 0.4 mg/mlstreptavidin Dynabeads (Dynal) was added to each of the standards andsamples at 50 μl/tube and incubated for 30 minutes at room temperature.Samples were then vortexed, and analyzed using an Origin 1.5 Analyzer(Igen) according to manufacturer's instructions.

This assay included 28 normal control samples and samples from 20patients diagnosed with SLE. Elevated levels of ZTNF4 were observed inthe serum of patients diagnosed with SLE, as compared with normalcontrol serum donors. ZTNF4 levels were calculated as a fold increase ofZTNF4 levels in the patient or control samples as compared to anarbitrary human reference serum sample. The average of the 28 controlsamples was 1.36 fold over the human reference sample and the average ofthe 20 SLE patient samples was 4.92. Seven out of the 20 SLE patientshad ZTNF4 levels that were two fold over the average of the controlsamples, whereas there was only one control individual that had agreater than two fold level over the control average.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention. Accordingly, the invention is notlimited except as by the appended claims.

1. A method for inhibiting the proliferation of tumor cells comprisingadministering to the tumor cells in need thereof a compositioncomprising a transmembrane activator and calcium modulator andcyclophilin ligand-interactor (TACI)-immunoglobulin fusion protein,wherein the TACI-immunoglobulin fusion protein comprises: (a) a TACIreceptor moiety that consists of: i) amino acid residues 30-110 of SEQID NO:2; or, ii) amino acid residues 30-154 of SEQ ID NO:2; wherein theTACI receptor moiety binds at least one of ZTNF2 or ZTNF4, and (b) animmunoglobulin moiety comprising a C_(H2) and C_(H3) domain.
 2. Themethod of claim 2, wherein the composition comprises a pharmaceuticalcomposition that comprises the TACI-immunoglobulin fusion protein and apharmaceutically acceptable carrier, and wherein the pharmaceuticalcomposition is administered to a mammalian subject which has a tumor. 3.The method of claim 2, wherein the administration of the pharmaceuticalcomposition inhibits the proliferation of B lymphocytes in the mammaliansubject.
 4. The method of claim 1, wherein the immunoglobulin moiety isan IgG1 immunoglobulin moiety.
 5. The method of claim 4, wherein theimmunoglobulin moiety is an IgG1 Fc fragment that comprises a disulfidelinked heavy chain hinge region, a C_(H2) domain and a C_(H3) domain. 6.The method of claim 1, wherein the TACI-immunoglobulin fusion proteinhas an amino acid sequence of SEQ ID NO:54.
 7. The method of claim 1,wherein the TACI-immunoglobulin fusion protein has an amino acidsequence comprising the secreted form of the amino acid sequence of SEQID NO:54.
 8. The method of claim 1, wherein the TACI-immunoglobulinfusion protein comprises the amino acid sequence of SEQ ID NO:54,wherein the optimized tPA (otPA) leader sequence (SEQ ID NO:25) has beenremoved.
 9. The method of claim 1, wherein the composition isadministered to cells cultured in vitro.
 10. The method of claim 1,wherein the TACI-immunoglobulin fusion protein is a dimer.
 11. A methodfor reducing circulating blood levels of ZTNF4 in a mammalian subjectcomprising administering to the mammalian subject a compositioncomprising a transmembrane activator and calcium modulator andcyclophilin ligand-interactor (TACI)-immunoglobulin fusion protein,wherein the TACI-immunoglobulin fusion protein comprises: (a) a TACIreceptor moiety that consists of i) amino acid residues 30-110 of SEQ IDNO:2; or, ii) amino acid residues 30-154 of SEQ ID NO:2; wherein theTACI receptor moiety binds ZTNF4, and (b) an immunoglobulin moietycomprising a C_(H2) and C_(H3) domain.
 12. The method of claim 11,wherein the immunoglobulin moiety is an IgG1 immunoglobulin moiety. 13.The method of claim 12, wherein the immunoglobulin moiety is an IgG1 Fcfragment that comprises a disulfide linked heavy chain hinge region, aC_(H2) domain and a C_(H3) domain.
 14. The method of claim 11, whereinthe TACI-immunoglobulin fusion protein has an amino acid sequence of SEQID NO:54.
 15. The method of claim 11, wherein the TACI-immunoglobulinfusion protein has an amino acid sequence comprising the secreted formof the amino acid sequence of SEQ ID NO:54.
 16. The method of claim 11,wherein the TACI-immunoglobulin fusion protein comprises the amino acidsequence of SEQ ID NO:54, wherein the optimized tPA (otPA) leadersequence (SEQ ID NO:25) has been removed.
 17. The method of claim 11,wherein the composition is administered to cells cultured in vitro. 18.The method of claim 11, wherein the TACI-immunoglobulin fusion proteinis a dimer.